WO2017081082A2 - Molécules d'acide nucléique optimisées - Google Patents

Molécules d'acide nucléique optimisées Download PDF

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WO2017081082A2
WO2017081082A2 PCT/EP2016/077145 EP2016077145W WO2017081082A2 WO 2017081082 A2 WO2017081082 A2 WO 2017081082A2 EP 2016077145 W EP2016077145 W EP 2016077145W WO 2017081082 A2 WO2017081082 A2 WO 2017081082A2
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nucleic acid
acid molecule
protein
utr
polypeptide
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PCT/EP2016/077145
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WO2017081082A3 (fr
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Patrick Baumhof
Susanne RAUCH
Aleksandra KOWALCZYK
Johannes Lutz
Edith JASNY
Benjamin Petsch
Andreas Thess
Thomas Schlake
Mariola Fotin-Mleczek
Regina HEIDENREICH
Sandra LAZZARO
Fatma DÖNER
Wolfgang Grosse
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Curevac Ag
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Priority to US15/774,423 priority Critical patent/US20180312545A1/en
Priority to EP16794593.0A priority patent/EP3374504A2/fr
Publication of WO2017081082A2 publication Critical patent/WO2017081082A2/fr
Publication of WO2017081082A3 publication Critical patent/WO2017081082A3/fr
Priority to US17/016,249 priority patent/US20200399322A1/en
Priority to US18/528,065 priority patent/US20240101608A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/145—Orthomyxoviridae, e.g. influenza virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/205—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Campylobacter (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55516—Proteins; Peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Optimized nucleic acid molecules The present invention concerns optimized nucleic acid molecules, methods for optimization of nucleic acid molecules and uses of optimized nucleic acid molecules, as well as biological entities comprising optimized nucleic acid molecules. Various aspects relating to optimization and to optimized nucleic acid molecules are subject of the present invention.
  • deoxyribonucleic acid and ribonucleic acid (RNA) are nucleic acid molecules which encode genetic information.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the encoded genetic information is translated into polypeptides and proteins by ribosomes.
  • In vitro translation can also be achieved in cell-free systems comprising ribosomes, and appropriate reagents.
  • RNA species encoding the genetic information for translation into polypeptides and proteins by ribosomes is called messenger RNA (mRNA).
  • mRNA messenger RNA
  • DNA is known to be relatively stable and easy to handle.
  • the use of DNA bears the risk of undesired insertion of the administered DNA-fragments into the target cell's or target subject's (patient's) genome, potentially resulting mutagenic events such as in loss of function of the impaired genes.
  • the undesired generation of anti-DNA antibodies has emerged.
  • RNA has been traditionally considered to be a rather unstable molecular species which may readi ly be degraded by ubiquitous RNAses.
  • RNA degradation contributes to the regulation of the RNA half- life time. That effect was considered and proven to fine tune the regulation of eukaryotic gene expression (Friedel et a/., 2009. conserveed principles of mammalian transcriptional regulation revealed by RNA half-life, Nucleic Acid Research 37(1 7): 1 -1 2). Accordingly, each naturally occurring mRNA has its individual half-life depending on the gene from which the mRNA is derived and in which cell type it is expressed. It contributes to the regulation of the expression level of this gene. Unstable RNAs are important to realize transient gene expression at distinct points in time. However, long-lived RNAs may be associated with accumulation of distinct proteins or continuous expression of genes.
  • the half-life of mRNAs may also be dependent on environmental factors, such as hormonal treatment, as has been shown, e.g., for insulin-like growth factor I, actin, and albumin mRNA (Johnson et ai, Newly synthesized RNA: Simultaneous measurement in intact cells of transcription rates and RNA stability of insulin-like growth factor I, actin, and albumin in growth hormone-stimulated hepatocytes, Proc. Natl. Acad. Sci., Vol. 88, pp. 5287-5291 , 1 991 ).
  • RNA For gene therapy and genetic vaccination, usually stable RNA is desired. This is, on the one hand, due to the fact that it is usually desired that the product encoded by the RNA sequence accumulates in vivo. On the other hand, the RNA has to maintain its structural and functional integrity when prepared for a suitable dosage form, in the course of its storage, and when administered. Thus, efforts were made to provide stable RNA molecules for gene therapy or genetic vaccination in order to prevent them from being subject to early degradation or decay.
  • nucleic acids comprising an increased amount of guanine (G) and/or cytosine (C) residues may be functionally more stable than nucleic acids containi ng a large amount of adenine (A) and thymine (T) or uraci l (U) nucleotides.
  • WO02/098443 provides a pharmaceutical composition containing an mRNA that is stabilised by sequence modifications in the coding region. Such a sequence modification takes advantage of the degeneracy of the genetic code.
  • RNA stabilization is limited by the provisions of the specific nucleotide sequence of each single RNA molecule which is not allowed to leave the space of the desired amino acid sequence. Also, that approach is restricted to coding regions of the RNA.
  • eukaryotic mRNA molecules contain characteristic stabi lising moieties.
  • they may comprise so-called untranslated regions (UTR) at their 5'-end (5'-UTR) and/or at their 3'- end (3'-UTR) as well as other structural features, such as a 5'-cap structure or a 3'-poly(A) tail.
  • UTR untranslated regions
  • 5'-UTR and 3'-UTR are typically transcribed from the genomic DNA and are, thus, a feature of the premature mRNA.
  • Characteristic structural features of mature mRNA such as the 5'-cap and the 3'-poly(A) tail (also called poly(A) tai l or poly(A) sequence) are usually added to the transcribed (premature) mRNA during mRNA processing.
  • a 3 '-poly(A) tail is typically a monotonous sequence stretch of adenosine nucleotides added to the 3 '-end of the transcribed mRNA. It may comprise up to about 400 adenosine nucleotides. It was found that the length of such a 3 '-poly(A) tail is potentially critical for the stability of individual mRNA.
  • RNA, 8, pp. 1 526-1 537, 2002 may be an important factor for the well-known stability of a-globin mRNA.
  • Rodgers et a/., Regulated a-globin mRNA decay is a cytoplasmic event proceeding through 3'-to-5' exosome-dependent decapping, RNA, 8, pp. 1 526-1 537, 2002).
  • the 3'-UTR of ⁇ -globin mRNA is apparently involved in the formation of a specific ribonucleoprotein-complex, the ⁇ -comp!ex, whose presence correlates with mRNA stability in vitro (Wang et a/., An mRNA stability complex functions with poly(A)-binding protein to stabilize mRNA in vitro, Molecular and Cellular biology, Vol 1 9, No. 7, July 1 999, p. 4552-4560).
  • ribosomal proteins which are typically transcribed in a constant manner so that some ribosomal protein mRNAs such as ribosomal protein S9 or ribosomal protein L32 are referred to as housekeeping genes (Janovick-Guretzky et al., Housekeeping Gene Expression in Bovine Liver is Affected by Physiological State, Feed Intake, and Dietary Treatment, J. Dairy Sci ., Vol. 90, 2007, p. 2246-2252).
  • the growth-associated expression pattern of ribosomal proteins is thus mainly due to regulation on the level of translation.
  • WO 2014/1 64253 A1 describes some specific nucleic acid molecules having 5'-UTRs and/or 3'-UTRs, without detail ing on translation efficiency of such molecules.
  • RNA molecules which are suitable for medical applications; particularly applications which involve the introduction of nucleic acids, such as DNA or RNA, into a subject's cell or tissue, followed by the translation of the information coded by the nucleic acids into the desired peptides or proteins.
  • beneficialal characteristics of mRNA were discovered in the recent years and clinical development of mRNA-based therapeutics is in progress (reviewed in Sahin et al. 201 4. Nat Rev Drug Discov. 201 4 Oct;1 3(1 0):759-80. doi: 1 0.1 038/nrd4278. Epub 2014 Sep 1 9; Kallen and Thess 2014. Ther Adv Vaccines. 201 4 Jan;2(1 ):10-31 . doi: 1 0.1 1 77/2051 01 3613508729. Review).
  • mRNA represents a transient copy of the coded genetic information in all organisms.
  • mRNA constructs may serve as a model for the synthesis of an unlimited variety of target proteins and, unlike DNA, represents all the necessary prerequisites for the preparation of a suitable vector for the transfer of exogenous genetic information in vivo.
  • nucleic acid molecules which may be suitable for application in gene therapy and/or genetic vaccination.
  • Another object of the present invention is to provide nucleic acid molecules coding for such a superior mRNA species which may be amenable for use in gene therapy and/or genetic vaccination.
  • the object underlying the present invention is solved by the claimed subject matter.
  • the inventors identified structural and functional aspects related to optimization of nucleic acid molecules, particularly RNA molecules. Such aspects are provided herein.
  • the invention also provides a modular system for combining aspects of RNA molecules, particularly optimized RNA molecules. The present invention therefore al lows the versati le combination of nucleic acid sequences, and thus provides numerous optimized RNA molecules based on the general principles disclosed herein.
  • mRNA constructs that may serve for i nformation carriers in protein therapies can be designed i n a way to obtain sufficient protein expression avoiding the activation of the immune system.
  • mRNA constructs that serve for information carriers in vaccination should be designed in a way to activate the immune system in the most efficient manner, that is e.g., to activate a strong cellular response for tumour vaccines or to induce a strong humoral response for prophylactic vaccines.
  • the adaptive immune response is typically understood to be an antigen-specific response of the immune system. Antigen specificity allows for the generation of responses that are tailored to specific pathogens or pathogen-infected cells. The ability to mount these tai lored responses is usually maintained in the body by "memory cells". Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it.
  • the first step of an adaptive immune response is the activation of naive antigen-specific T cells or different immune cells able to induce an antigen-specific immune response by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing.
  • dendritic cells The three cell types that may serve as antigen-presenting cells are dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses.
  • Dendritic cells may take up antigens by phagocytosis and macropinocytosis and may become stimulated by contact with e.g. a foreign antigen to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells.
  • Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express MHC molecules.
  • the unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells.
  • MHC-molecules are, typically, responsible for presentation of an antigen to T-cells. Therein, presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells.
  • effector T cells The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Thl cells to produce different classes of antibody, thus driving the humoral immune response.
  • T cells recognize an antigen by their T cell receptors which do not recognize and bind the antigen directly, but instead recognize short peptide fragments e.g. of pathogen-derived protein antigens, e.g. so-called epitopes, which are bound to MHC molecules on the surfaces of other cells.
  • the adaptive immune system is essentially dedicated to eliminate or prevent pathogenic growth. It typically regulates the adaptive immune response by providing the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered.
  • the system is highly adaptable because of somatic hypermutation (a process of accelerated somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte.
  • Adjuvant/adjuvant component in the broadest sense is typically a pharmacological and/or immunological agent that may modify, e.g. enhance, the effect of other agents, such as a drug or vaccine. It is to be interpreted in a broad sense and refers to a broad spectrum of substances. Typically, these substances are able to increase the immunogenicity of antigens.
  • adjuvants may be recognized by the innate immune systems and, e.g., may elicit an innate immune response. "Adjuvants" typically do not elicit an adaptive immune response. Insofar, "adjuvants" do not qualify as antigens. Their mode of action is distinct from the effects triggered by antigens resulting in an adaptive immune response.
  • Antigen refers typically to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, e.g. by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response.
  • an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells.
  • an antigen may be the product of translation of a provided nucleic acid molecule, preferably an mRNA as defined herein.
  • fragments, variants and derivatives of peptides and proteins comprising at least one epitope are understood as antigens.
  • tumour antigens and pathogenic antigens as defined herein are particularly preferred.
  • An artificial nucleic acid molecule may typically be understood to be a nucleic acid molecule - e.g. DNA or RNA - that does not occur naturally.
  • an artificial nucleic acid molecule may be understood as a non-natural nucleic acid molecule.
  • Such nucleic acid molecule may be non-natural due to its individual sequence (which does not occur naturally) and/or due to other modifications, e.g. structural modifications of nucleotides which do not occur naturally.
  • An artificial nucleic acid molecule may be a DNA molecule, an RNA molecule or a hybrid-molecule comprising DNA and RNA portions.
  • artificial nucleic acid molecules may be designed and/or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence).
  • an artificial sequence is usually a sequence that may not occur naturally, i.e. it differs from the wild-type sequence by at least one nucleotide.
  • wild-type may be understood as a sequence occurring in nature.
  • artificial nucleic acid molecule is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.
  • Bicistronic RNA, multicistronic RNA A bicistronic or multicistronic RNA is typically an RNA, preferably an mRNA that typically may have two (bicistronic) or more (multicistronic) open reading frames (ORF).
  • An open reading frame in this context is a sequence of codons that is translatable into a peptide or protein.
  • Carrier / polymeric carrier A carrier in the context of the invention is any compound that faci litates transport and/or complexation of another compound. Said other compound can be referred to as "cargo".
  • a polymeric carrier is typically a carrier that is formed of a polymeric molecule.
  • a carrier may be associated to its cargo by covalent or non-covalent interaction.
  • a carrier may transport nucleic acids, e.g. RNA or DNA, to the target cells.
  • the carrier may - in some embodiments - be a cationic component.
  • Cationic component typically refers to a charged molecule, which is positively charged (cation) at a pH value typically from 1 to 9, preferably at a pH value of or below 9 (e.g. from 5 to 9), of or below 8 (e.g. from 5 to 8), of or below 7 (e.g. from 5 to 7), most preferably at a physiological pH, e.g. from 7.3 to 7.4.
  • a cationic component may be any positively charged compound or polymer, preferably a cationic peptide or protein which is positively charged under physiological conditions, particularly under physiological conditions in vivo.
  • a "cationic peptide or protein” may contain at least one positively charged amino acid, or more than one positively charged amino acid, e.g.
  • a 5 '-cap is an entity, typically a modified nucleotide entity, which generally "caps" the 5'-end of a mature mRNA.
  • a 5 '-cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide.
  • the 5'-cap is linked to the 5'- terminus via a 5'-5'-triphosphate linkage.
  • a 5'-cap may be methylated, e.g.
  • N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap, typically the 5'-end of an RNA.
  • 5'cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, l -(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3', 4'- seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'
  • Cellular immunity relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen.
  • cellular immunity is not based on antibodies, but on the activation of cells of the immune system.
  • a cellular immune response may be characterized e.g. by activating antigen- specific cytotoxic T-lymphocytes that are able to induce apoptosis in cel ls, e.g. specific immune cells l ike dendritic cells or other cells, displaying epitopes of foreign antigens on their surface.
  • Such cells may be virus-infected or infected with intracellular bacteria, or cancer cel ls displaying tumor antigens. Further characteristics may be activation of macrophages and natural killer cells, enabling them to destroy pathogens and stimulation of cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.
  • DNA is the usual abbreviation for deoxy-ribonucleic acid. It is a nucleic acid molecule, i .e. a polymer consisting of nucleotides. These nucleotides are usually deoxy- adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosi ne- monophosphate and deoxy-cytidine-monophosphate monomers which are - by themselves - composed of a sugar moiety (deoxyribose), a base moiety and a phosphate moiety, and polymerise by a characteristic backbone structure.
  • the backbone structure is, typically, formed by phosphodiester bonds between the sugar moiety of the nucleotide, i .e. deoxyribose, of a first and a phosphate moiety of a second, adjacent monomer.
  • the specific order of the monomers i.e. the order of the bases linked to the sugar/phosphate-backbone, is called the DNA sequence.
  • DNA may be single stranded or double stranded. In the double stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g. by A/T-base-pairing and G/C-base-pairing.
  • Element An element, as used herein, generally refers to a polypeptide sub-sequence. Typically, more than one polypeptide sub-sequences are arranged in linear order, so that several same or different sub-sequences or elements are typically present in a polypeptide sequence. Without limiting the technical content, the term “element” is used herein to refer to a module on polypeptide or protein level. This use reflects the general use in the art for polypeptide or protein elements, as i llustrated e.g. by the well-known term "transmembrane element".
  • element is not limited to those polypeptide or protein modules that have been termed “element” in the prior art, but generally refers to a polypeptide or protein sub-sequence or module, as defined herein.
  • an element is encoded by a nucleic acid module (moiety), as defined herein.
  • T cel l epitopes or parts of the proteins in the context of the present invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 1 1 , or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 1 3 or more amino acids, e.g.
  • fragments may be selected from any part of the amino acid sequence.
  • T cells typically recognized by T cells in form of a complex consisting of the peptide fragment and an MHC molecule, i .e. the fragments are typically not recognized in their native form.
  • B cell epitopes are typical ly fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably having 5 to 1 5 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form.
  • Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides.
  • antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.
  • a fragment of a sequence may typically be a shorter portion of a full-length sequence of e.g. a nucleic acid molecule or an amino acid sequence. Accordingly, a fragment, typically, consists of a sequence that is identical to the corresponding stretch within the full-length sequence.
  • a preferred fragment of a sequence in the context of the present invention consists of a continuous stretch of entities, such as nucleotides or amino acids corresponding to a continuous stretch of entities in the molecule the fragment is derived from, which represents at least 5%, 1 0%, 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the total (i.e. full-length) molecule from which the fragment is derived.
  • a G/C-modified nucleic acid may typically be a nucleic acid, preferably an artificial nucleic acid molecule as defined herein, based on a modified wi ld-type sequence comprising a preferably increased number of guanosine and/or cytosine nucleotides as compared to the wild-type sequence. Such an increased number may be generated by substitution of codons containing adenosine or thymidine nucleotides by codons containing guanosine or cytosine nucleotides. If the enriched G/C content occurs in a coding region of DNA or RNA, it makes use of the degeneracy of the genetic code.
  • the codon substitutions preferably do not alter the encoded amino acid residues, but exclusively increase the G/C content of the nucleic acid molecule.
  • An artificial nucleic acid molecule which is G/C modified and which therefore exhibits at least one superior property with respect to a non-G/C optimized nucleic acid molecule encoding the same polypeptide, is termed "optimized nucleic acid molecule”.
  • Gene therapy may typically be understood to mean a treatment of a patient's body or isolated elements of a patient's body, for example isolated tissues/cells, by nucleic acids encoding a peptide or protein. It typically may comprise at least one of the steps of a) administration of a nucleic acid, preferably an optimized nucleic acid molecule as defined herein, directly to the patient - by any suitable administration route - or in vitro to isolated cells/tissues of the patient, which results in transfection of the patient's cells either in vivo/ex vivo or in vitro; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re-administration of isolated, transfected cells to the patient, if the nucleic acid has not been administered directly to the patient.
  • a nucleic acid preferably an optimized nucleic acid molecule as defined herein
  • Genetic vaccination may typically be understood to be vaccination by administration of a nucleic acid molecule encoding an antigen or an immunogen or fragments thereof.
  • the nucleic acid molecule may be administered to a subject's body or to isolated cells of a subject. Upon transfection of certain cells of the body or upon transfection of the isolated cells, the antigen or immunogen may be expressed by those cells and subsequently presented to the immune system, eliciting an adaptive, i.e. antigen-specific immune response.
  • genetic vaccination typically comprises at least one of the steps of a) administration of a nucleic acid, preferably an optimized nucleic acid molecule as defi ned herein, to a subject, preferably a patient, or to isolated cells of a subject, preferably a patient, which usually results in transfection of the subject's cells either in vivo or in vitro; b) transcription and/or translation of the introduced nucleic acid molecule; and optionally c) re- administration of isolated, transfected cells to the subject, preferably the patient, if the nucleic acid has not been administered directly to the patient.
  • Heterologous sequence Two sequences are typically understood to be 'heterologous' if they are not derivable from the same gene. I.e., although heterologous sequences may be derivable from the same organism, they naturally (in nature) do not occur in the same nucleic acid molecule, such as in the same mRNA.
  • Humoral immunity refers typically to antibody production and optionally to accessory processes accompanying antibody production.
  • a humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal centre formation and isotype switching, affinity maturation and memory cell generation.
  • Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.
  • Immunogen In the context of the present invention an immunogen may be typically understood to be a compound that is able to stimulate an immune response.
  • an immunogen is a peptide, polypeptide, or protein.
  • an immunogen in the sense of the present invention is the product of translation of a provided nucleic acid molecule, preferably an optimized nucleic acid molecule as defined herein.
  • an immunogen elicits at least an adaptive immune response.
  • an immunostimulatory composition may be typically understood to be a composition containing at least one component which is able to induce an immune response or from which a component which is able to induce an immune response is derivable. Such immune response may be preferably an i nnate immune response or a combination of an adaptive and an innate immune response.
  • an immunostimulatory composition in the context of the invention contains at least one optimized nucleic acid molecule, more preferably an RNA, for example an mRNA molecule.
  • the immunostimulatory component, such as the mRNA may be complexed with a suitable carrier.
  • the immunostimulatory composition may comprise an mRNA/carrier- complex.
  • the immunostimulatory composition may comprise an adjuvant and/or a suitable vehicle for the immunostimulatory component, such as the mRNA.
  • Immune response An immune response may typically be a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response), or a combination thereof.
  • the immune system may protect organisms from infection. If a pathogen succeeds in passing a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and al lows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. According to this, the immune system comprises the innate and the adaptive immune system. Each of these two parts typically contains so called humoral and cellular components.
  • Immunostimulatory RNA in the context of the invention may typically be an RNA that is able to induce an innate immune response. It usually does not have an open reading frame and thus does not provide a peptide-antigen or immunogen but elicits an immune response e.g. by binding to a specific kind of Toll-like-receptor (TLR) or other suitable receptors.
  • TLR Toll-like-receptor
  • mRNAs having an open reading frame and coding for a peptide/protein may induce an innate immune response and, thus, may be immunostimulatory RNAs.
  • the innate immune system also known as non-specific (or unspecific) immune system, typically comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system may recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host.
  • the innate immune system may be, e.g., activated by ligands of Toll-like receptors (TLRs) or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1 1 , IL-12, IL-13, IL-14, IL-15, IL-1 6, IL-1 7, IL-18, IL-19, IL-20, IL-21 , IL- 22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31 , IL-32, IL-33, IFN-alpha, IFN- beta, IFN-gamma, GM-CSF,
  • the pharmaceutical composition according to the present invention may comprise one or more such substances.
  • a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system; and/or acting as a physical and chemical barrier to infectious agents.
  • a cloning site is typically understood to be a segment of a nucleic acid molecule, which is suitable for insertion of a nucleic acid sequence, e.g., a nucleic acid sequence comprising an open reading frame. Insertion may be performed by any molecular biological method known to the one skilled in the art, e.g. by restriction and ligation.
  • a cloning site typically comprises one or more restriction enzyme recognition sites (restriction sites). These one or more restrictions sites may be recognized by restriction enzymes which cleave the DNA at these sites.
  • a cloning site which comprises more than one restriction site may also be termed a multiple cloning site (MCS) or a polylinker.
  • MCS multiple cloning site
  • a module generally refers to a polypeptide sub-sequence or a polynucleotide sub-sequence.
  • a sub-sequence is a sequence forming part of a sequence.
  • a modular design principle is provided that is suitable to generate a polypeptide sequence or a polynucleotide sequence comprising several (more than one) subsequences or modules. Typically, the respective sub-sequences are arranged in linear order. Thus, several same or different sub-sequences or modules are typically present in a polypeptide sequence or polynucleotide sequence, respectively.
  • the term "moiety” is used herein to refer to a module on nucleic acid level
  • the term “element” is used herein to refer to a module on polypeptide or protein level.
  • a moiety generally refers to a polynucleotide sub-sequence. Typically, more than one polynucleotide sub-sequences are arranged in linear order, so that several same or different sub-sequences or moieties are typically present in a polynucleotide sequence.
  • the term "moiety” is used herein to refer to a module on nucleic acid level. This is reflects the use of this term e.g. in the area of combinatorial chemistry, where said term is generally used to refer to one of the portions into which a given molecule can be (e.g. mentally) divided.
  • the term moiety refers to a portion of a nucleic acid molecule; the nucleic acid molecule can be (e.g. mentally) divided into several moieties.
  • a moiety may encode a polypeptide or protein module (element), as defined herein, or may be a non-coding moiety.
  • Nucleic acid molecule is a molecule comprising, preferably consisting of nucleic acid components.
  • the term nucleic acid molecule preferably refers to DNA or RNA molecules. It is preferably used synonymous with the term "polynucleotide".
  • a nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers which are covalently linked to each other by phosphodiester-bonds of a sugar/phosphate-backbone.
  • the term "nucleic acid molecule” also encompasses modified nucleic acid molecules, such as base-modified, sugar-modified or backbone-modified etc. DNA or RNA molecules.
  • Open reading frame in the context of the invention may typically be a sequence of several nucleotide triplets which may be translated into a peptide or protein.
  • An open reading frame preferably contains a start codon, i.e. a combination of three subsequent nucleotides coding usually for the amino acid methionine (ATG), at its 5'- end and a subsequent region which usually exhibits a length which is a multiple of 3 nucleotides.
  • An ORF is preferably terminated by a stop-codon (e.g., TAA, TAG, and TGA). Typically, this is the only stop-codon of the open reading frame.
  • an open reading frame i n the context of the present invention is preferably a nucleotide sequence, consisting of a number of nucleotides that may be divided by three, which starts with a start codon (e.g. ATG) and which preferably terminates with a stop codon (e.g., TAA, TGA, or TAG).
  • the open reading frame may be isolated or it may be incorporated in a longer nucleic acid sequence, for example in a vector or an mRNA.
  • An open reading frame may also be termed "protein coding region" or "coding sequence" (cds).
  • Optimized nucleic acid molecule In general, an optimized nucleic acid molecule is a nucleic acid molecule not found in nature. In other words, it is an artificial nucleic acid molecule, i.e. not a wild-type nucleic acid molecule.
  • the nucleic acid molecule of the present invention is distinguished from a wild-type nucleic acid molecule by at least one structural feature. The distinguishing structural feature is selected from sequence modifications and base modifications. As described herein below, a sequence modification alters the polynucleotide sequence with respect to a wi ld-type nucleic acid molecule.
  • sequence modification is typically selected among an addition, a deletion, an insertion and a substitution of one or more nucleic acid residues, with respect to a wi ld-type nucleic acid molecule. More than one such sequence modifications can be present in an optimized nucleic acid molecule.
  • the optimized nucleic acid molecules of the present invention allow for the versatile combination of multiple polypeptide or protein elements, encoded by respective nucleic acid moieties.
  • the optimized nucleic molecule of the present invention is characterized by at least one addition of one or more nucleic acid residues, in practice, addition of at least one nucleic acid moiety (coding or non-coding), as described herein.
  • Base modification means that at least one base of a nucleic acid or (deoxyribonucleic acid or ribonucleic acid) is altered.
  • the at least one distinguishing structural feature provides - or contributes to - a functional property of the optimized nucleic acid molecule which is not exhibited by the non- optimized (wi ld-type) nucleic acid molecule.
  • Peptide A peptide or oligopeptide or polypeptide is typically a polymer of at least two amino acid monomers, linked by peptide bonds.
  • An oligopeptide typically contains less than 50 monomer units, although the term peptide or oligopeptide is not a disclaimer for molecules having more than 50 monomer units.
  • Polypeptides typically have between 50 and 600 monomer units, although the term polypeptide is neither a disclaimer for molecules having more than 600 monomer units, nor for molecules havi ng less than 50 monomer units.
  • Large peptides, i.e. peptides typically having more than 50 monomer units, or even more than 600 monomer units, are also referred to as proteins.
  • a pharmaceutically effective amount in the context of the invention is typically understood to be an amount that is sufficient to induce a pharmaceutical effect, such as an immune response, altering a pathological level of an expressed peptide or protein, or substituting a lacking gene product, e.g., in case of a pathological situation.
  • a protein typically comprises one or more peptides or polypeptides. A protein is typically folded into 3-dimensional form, which may be required for the protein to exert its biological function.
  • Poly(A) sequence A poIy(A) sequence, also called poly(A) tai l or 3 '-poly(A) tail, is typically understood to be a sequence of adenosine nucleotides, e.g., of up to about 400 adenosine nucleotides, e.g. from about 20 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenosine nucleotides.
  • a poly(A) sequence is typically located at the 3'end of an mRNA.
  • a poly(A) sequence may be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector.
  • Polyadenylation is typical ly understood to be the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule, e.g. to a premature mRNA. Polyadenylation may be induced by a so cal led polyadenylation signal. This signal is preferably located within a stretch of nucleotides at the 3'-end of a nucleic acid molecule, such as an RNA molecule, to be polyadenylated.
  • a polyadenylation signal typically comprises a hexamer consisting of adenine and uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA.
  • RNA maturation from pre-mRNA to mature mRNA comprises the step of polyadenylation.
  • restriction site also termed restriction enzyme recognition site, is a nucleotide sequence recognized by a restriction enzyme.
  • a restriction site is typically a short, preferably palindromic nucleotide sequence, e.g. a sequence comprising 4 to 8 nucleotides.
  • a restriction site is preferably specifically recognized by a restriction enzyme.
  • the restriction enzyme typically cleaves a nucleotide sequence comprising a restriction site at this site.
  • the restriction enzyme typically cuts both strands of the nucleotide sequence.
  • RNA is the usual abbreviation for ribonucleic acid. It is a nucleic acid molecule, i.e. a polymer consisting of nucleotides. These nucleotides are usually adenosine- monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine- monophosphate monomers which are connected to each other along a so-cal led backbone. The backbone is formed by phosphodiester bonds between the sugar, i.e. ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific succession of the monomers is called the RNA-sequence.
  • RNA may be obtainable by transcription of a DNA- sequence, e.g., inside a cell.
  • transcription is typical ly performed inside the nucleus or the mitochondria.
  • transcription of DNA usually results in the so- cal led premature RNA which has to be processed into so-called messenger-RNA, usually abbreviated as mRNA.
  • Processing of the premature RNA e.g. in eukaryotic organisms, comprises a variety of different posttranscriptional-modifications such as splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA.
  • the mature messenger RNA usual ly provides the nucleotide sequence that may be translated into an amino-acid sequence of a particular peptide or protein.
  • a mature mRNA comprises a 5'-cap, a 5'-UTR, an open reading frame, a 3'-UTR and a poly(A) sequence.
  • Sequence of a nucleic acid molecule The sequence of a nucleic acid molecule is typically understood to be the particular and individual order, i.e. the succession of its nucleotides.
  • sequence of a protein or peptide is typically understood to be the order, i.e. the succession of its amino acids.
  • Sequence identity Two or more sequences are identical if they exhibit the same length and order of nucleotides or amino acids.
  • the percentage of identity typically describes the extent to which two sequences are identical, i.e. it typically describes the percentage of nucleotides that correspond in their sequence position with identical nucleotides of a reference-sequence.
  • the sequences to be compared are considered to exhibit the same length, i.e. the length of the longest sequence of the sequences to be compared. This means that a first sequence consisting of 8 nucleotides is 80% identical to a second sequence consisting of 1 0 nucleotides comprising the first sequence.
  • identity of sequences preferably relates to the percentage of nucleotides of a sequence which have the same position in two or more sequences having the same length. Gaps are usually regarded as non-identical positions, irrespective of their actual position in an alignment.
  • a stabilized nucleic acid molecule is a nucleic acid molecule, preferably a DNA or RNA molecule that is modified such, that it is more stable to disintegration or degradation, e.g., by environmental factors or enzymatic digest, such as by an exo- or endonuclease degradation, than the nucleic acid molecule without the modification.
  • a stabilized nucleic acid molecule in the context of the present invention is stabi lized in a cell, such as a prokaryotic or eukaryotic cell, preferably in a mammalian cell, such as a human cell.
  • the stabilization effect may also be exerted outside of cells, e.g. in a buffer solution etc., for example, in a manufacturing process for a pharmaceutical composition comprising the stabilized nucleic acid molecule.
  • Transfection refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably into eukaryotic cells.
  • nucleic acid molecules such as DNA or RNA (e.g. mRNA) molecules
  • transfection encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, preferably into eukaryotic cells, such as into mammalian cells. Such methods encompass, for example, electroporation, l ipofection, e.g.
  • the introduction is non-viral.
  • a vaccine is typical ly understood to be a prophylactic or therapeutic material providing at least one antigen, preferably an immunogen.
  • the antigen or immunogen may be derived from any material that is suitable for vaccination.
  • the antigen or immunogen may be derived from a pathogen, such as from bacteria or virus particles etc., or from a tumor or cancerous tissue.
  • the antigen or immunogen stimulates the body's adaptive immune system to provide an adaptive immune response.
  • Vector refers to a nucleic acid molecule, preferably to an artificial nucleic acid molecule.
  • a vector in the context of the present invention is suitable for incorporating or harbouring a desired nucleic acid sequence, such as preferably an optimized nucleic acid molecule as described herein, comprising at least one open reading frame (ORF).
  • Such vectors may be storage vectors, expression vectors, cloning vectors, transfer vectors etc.
  • a storage vector is a vector which allows the convenient storage of a nucleic acid molecule, for example, of an mRNA molecule.
  • the vector may comprise a sequence correspondi ng, e.g., to a desired mRNA sequence or a part thereof, such as a sequence corresponding to the open reading frame and the 3'-UTR and/or the 5'-UTR of an mRNA.
  • An expression vector may be used for production of expression products such as RNA, e.g. mRNA, or peptides, polypeptides or proteins.
  • an expression vector may comprise sequences needed for transcription of a sequence stretch of the vector, such as a promoter sequence, e.g. an RNA polymerase promoter sequence.
  • a cloning vector is typically a vector that contains a cloning site, which may be used to incorporate nucleic acid sequences into the vector.
  • a cloning vector may be, e.g., a plasmid vector or a bacteriophage vector.
  • a transfer vector may be a vector which is suitable for transferring nucleic acid molecules into cells or organisms, for example, viral vectors.
  • a vector in the context of the present invention may be, e.g., an RNA vector or a DNA vector.
  • a vector is a DNA molecule.
  • a vector in the sense of the present invention comprises a cloning site, a selection marker, such as an antibiotic resistance factor, and a sequence suitable for multiplication of the vector, such as an origin of replication.
  • a vector in the context of the present invention is a plasmid vector.
  • a vehicle is typically understood to be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound.
  • a compound such as a pharmaceutically active compound.
  • it may be a physiologically acceptable liquid which is suitable for storing, transporting, and/or administering a pharmaceutically active compound.
  • 3'-untranslated region 3'-UTR: Generally, the term “3'-UTR” refers to a part of the artificial nucleic acid molecule of the invention, which is located 3' (i.e. "downstream") of an open reading frame and which is not translated into protein. Typically, a 3'-UTR is the part of an mRNA which is located between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of the mRNA. In the context of the present invention, a 3'-UTR is suitably comprised in the optimized nucleic acid molecule.
  • ORF open reading frame
  • CDS coding sequence
  • 3'-UTR may also comprise moieties, which are not encoded in the template, from which an RNA is transcribed, but which are added after transcription during maturation, e.g. a poly(A) sequence.
  • a 3'-UTR of the mRNA is not translated into an amino acid sequence.
  • the 3'-UTR sequence is generally encoded by the gene which is transcribed into the respective mRNA during the gene expression process.
  • the genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns.
  • the pre-mature mRNA is then further processed into mature mRNA in a maturation process.
  • This maturation process comprises the steps of 5'capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3'-end, such as polyadenylation of the 3'-end of the pre-mature mRNA and optional endo-/ or exonuclease cleavages etc.
  • a 3'-UTR corresponds to the sequence of a mature mRNA which is located between the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and the poly(A) sequence of the mRNA.
  • the 3'-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3'- UTR sequence, or a DNA sequence which corresponds to such RNA sequence.
  • a 3'-UTR of a gene is the sequence which corresponds to the 3'-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term "3'-UTR of a gene” encompasses the DNA sequence and the RNA sequence (both sense and antisense strand and both mature and immature) of the 3'-UTR.
  • the 3'UTRs have a length of more than 20, 30, 40 or 50 nucleotides.
  • 5'-untranslated region 5'-UTR: Generally, the term “5'-UTR” refers to a part of the artificial nucleic acid molecule, which is located 5' (i.e. "upstream") of an open reading frame (ORF) and which is not translated into protein.
  • a 5'-UTR is typically understood to be a particular section of messenger RNA (mRNA), which is located 5' of the open reading frame of the mRNA.
  • mRNA messenger RNA
  • a 5'-UTR is preferably present 5' of an open reading frame encoding a polypeptide comprising a polypeptide of interest.
  • the 5'- UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame.
  • the 5'UTRs have a length of more than 20, 30, 40 or 50 nucleotides.
  • the 5'-UTR may comprise moieties for controlling gene expression, also called regulatory moieties. Such regulatory moieties may be, for example, ribosomal binding sites.
  • the 5'-UTR may be posttranscriptionally modified, for example by addition of a 5'-CAP.
  • a 5'-UTR of the mRNA is not translated into an amino acid sequence.
  • the 5'-UTR sequence is generally encoded by the gene which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns.
  • the pre-mature mRNA is then further processed into mature mRNA in a maturation process.
  • This maturation process comprises the steps of 5'capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3'-end, such as polyadenylation of the 3'-end of the pre-mature mRNA and optional endo-/ or exonuclease cleavages etc..
  • a 5'-UTR corresponds to the sequence of a mature mRNA which is located between the start codon and, for example, the 5'-CAP.
  • the 5'-UTR corresponds to the sequence which extends from a nucleotide located 3' to the 5'-CAP, more preferably from the nucleotide located immediately 3' to the 5'-CAP, to a nucleotide located 5' to the start codon of the protein coding region, preferably to the nucleotide located immediately 5' to the start codon of the protein coding region.
  • the nucleotide located immediately 3' to the 5'-CAP of a mature mRNA typically corresponds to the transcriptional start site.
  • the term “corresponds to” means that the 5'-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 5'-UTR sequence, or a DNA sequence which corresponds to such RNA sequence.
  • a 5'-UTR of a gene is the sequence which corresponds to the 5'-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA.
  • the term “5'-UTR of a gene” encompasses the DNA sequence and the RNA sequence (both sense and antisense strand and both mature and immature) of the 5'-UTR.
  • TOP 5'Terminal Oligopyrimidine Tract
  • the 5'terminal oligopyrimidine tract (TOP) is typically a stretch of pyrimidine nucleotides located in the 5' terminal region of a nucleic acid molecule, such as the 5' terminal region of certain mRNA molecules or the 5' terminal region of a functional entity, e.g. the transcribed region, of certain genes.
  • the sequence starts with a cytidine, which usual ly corresponds to the transcriptional start site, and is followed by a stretch of usually about 3 to 30 pyrimidine nucleotides.
  • the TOP may comprise 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 1 2, 1 3, 14, 1 5, 1 6, 1 7, 1 8, 1 9, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides.
  • Messenger RNA that contains a 5 'terminal oligopyrimidine tract is often referred to as TOP mRNA. Accordingly, genes that provide such messenger RNAs are referred to as TOP genes.
  • TOP sequences have, for example, been found in genes and mRNAs encoding peptide elongation factors and ribosomal proteins.
  • TOP motif In the context of the present invention, a TOP motif is a nucleic acid sequence which corresponds to a 5'TOP as defined above. Thus, a TOP motif in the context of the present invention is preferably a stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.
  • the TOP-motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, most preferably at least 8 pyrimidi ne nucleotides, wherein the stretch of pyrimidi ne nucleotides preferably starts at its 5'end with a cytosine nucleotide.
  • the TOP-motif preferably starts at its 5'end with the transcriptional start site and ends one nucleotide 5' to the first purin residue in said gene or mRNA.
  • a TOP motif in the sense of the present invention is preferably located at the 5'end of a sequence which represents a 5'-UTR or at the 5'end of a sequence which codes for a 5'-UTR.
  • TOP motif a stretch of 3 or more pyrimidine nucleotides is called "TOP motif" in the sense of the present invention if this stretch is located at the 5'end of a respective sequence, such as an artificial nucleic acid molecule (e.g. the optimized nucleic acid molecule of the present invention), the 5'-UTR moiety of said artificial nucleic acid molecule, or the nucleic acid sequence which is derived from the 5'-UTR of a TOP gene as described herein.
  • an artificial nucleic acid molecule e.g. the optimized nucleic acid molecule of the present invention
  • the 5'-UTR moiety of said artificial nucleic acid molecule or the nucleic acid sequence which is derived from the 5'-UTR of a TOP gene as described herein.
  • TOP gene TOP genes are typically characterised by the presence of a 5' terminal oligopyrimidine tract. Furthermore, most TOP genes are characterized by a growth-associated translational regulation. However, also TOP genes with a tissue specific translational regulation are known.
  • the 5'-UTR of a TOP gene corresponds to the sequence of a 5'-UTR of a mature mRNA derived from a TOP gene, which preferably extends from the nucleotide located 3' to the 5'-CAP to the nucleotide located 5' to the start codon.
  • a 5'-UTR of a TOP gene typically does not comprise any start codons, preferably no upstream AUGs (uAUGs) or upstream open reading frames (uORFs).
  • upstream AUGs and upstream open reading frames are typically understood to be AUGs and open reading frames that occur 5' of the start codon (AUG) of the open reading frame that should be translated.
  • the 5'-UTRs of TOP genes are generally rather short.
  • the lengths of 5'-UTRs of TOP genes may vary between 20 nucleotides up to 500 nucleotides, and are typically less than about 200 nucleotides, preferably less than about 1 50 nucleotides, more preferably less than about 1 00 nucleotides.
  • Exemplary 5'-UTRs of TOP genes in the sense of the present invention are the nucleic acid sequences extending from the nucleotide at position 5 to the nucleotide located immediately 5' to the start codon (e.g. the ATG) in the sequences according to SEQ ID NOs. 1 -1 363 of the patent application WO2013/1 43700, whose disclosure is incorporated herewith by reference.
  • a particularly preferred fragment of a 5'-UTR of a TOP gene is a 5'-UTR of a TOP gene lacking the 5'TOP motif.
  • the terms "5'-UTR of a TOP gene” or “5'-TOP UTR” preferably refer to the 5'-UTR of a natural ly occurring TOP gene.
  • a preferred example is represented by SEQ ID NO: 1 674 (5'-UTR of human ribosomal protein Large 32 lacking the 5' terminal oligopyrimidine tract); corresponding to SEQ ID NO. 1 368 of the patent application WO201 3/1 43700).
  • Wild-type e.g. wild-type nucleic acid molecule
  • wild-type nucleic acid molecule may typically be understood to be a nucleic acid molecule - e.g. a DNA or an RNA - that occurs naturally.
  • a wild-type nucleic acid molecule may be understood as a natural nucleic acid molecule.
  • Such nucleic acid molecule may be natural due to its individual sequence (which occurs naturally) and/or due to other modifications, e.g. structural modifications of nucleotides which occur natural ly.
  • a wild-type nucleic acid molecule may be a DNA molecule, an RNA molecule or a hybrid-molecule comprising DNA and RNA portions.
  • wild- type refers to any sequence as long as it occurs in nature, reflection in publically accessible sequence collections such as GenBank is not required.
  • NASH National Institute of Health
  • GenBank accessible through the NCBI Entrez retrieval system: http://www.ncbi.nlm.nih.gov), (Nucleic Acids Research, 201 3; 4 ⁇ (D1 ):D36-42), including publicly available wi ld-type sequences.
  • Each GenBank record is assigned a unique constant identifier called an accession number and appears on the ACCESSION line of a GenBank record; and changes to the sequence data are tracked by an integer extension of the accession number which appears on the VERSION line of the GenBank record.
  • wild- type nucleic acid molecule is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.
  • the optimized nucleic acid molecules of the present invention are preferably not wild-type nucleic acid molecules.
  • the present invention concerns optimized nucleic acid molecules, methods for optimization of nucleic acid molecules and uses of optimized nucleic acid molecules, as well as biological entities comprising optimized nucleic acid molecules.
  • an optimized nucleic acid molecule is a nucleic acid molecule not found in nature. In other words, it is an artificial nucleic acid molecule, i.e. not a wild-type nucleic acid molecule.
  • an optimized nucleic acid molecule of the present invention is superior to a naturally occurring nucleic acid molecule.
  • Various aspects relating to optimization are subject of the present invention, as detailed herein.
  • the nucleic acid molecule of the present invention is distinguished from a wild-type nucleic acid molecule by at least one structural feature.
  • the distinguishing structural feature is selected from sequence features (addition, deletion, insertion and/or substitution of one or more nucleotide, with respect to a wild-type nucleic acid molecule) and nucleoside modifications (altered natural or non-natural nucleotide in at least one position).
  • sequence features addition, deletion, insertion and/or substitution of one or more nucleotide, with respect to a wild-type nucleic acid molecule
  • nucleoside modifications altered natural or non-natural nucleotide in at least one position.
  • a modular design principle is provided that is suitable to generate an mRNA construct tailored for a respective medical application.
  • the optimized nucleic acid molecules of the present invention allow for the versatile combination of multiple moieties or elements.
  • the present invention relates to a nucleic acid molecule comprising at least two modules, and wherein at least one module is an open reading frame (ORF) encoding a polypeptide or protein of interest, and wherein at least one module is selected from (i) a further module encoding a polypeptide or protein element (coding module) and (i i) a module not encoding a polypeptide or protein element (non-coding module).
  • ORF open reading frame
  • the at least two modules can be numbered, e.g. first module, second module...
  • a first module can be an open reading frame (ORF) encoding a polypeptide or protei n of interest
  • a second module can be selected from (i) a further module encoding a polypeptide or protein element (coding module) and (ii) a module not encoding a polypeptide or protein element (non-coding module).
  • the terms first and second do not imply a specific arrangement; thus, the first module can be located either 5' Upstream) or 3' (downstream) of the second module.
  • Each module is a nucleic acid moiety.
  • nucleic acid molecule comprises multiple (two or more) moieties, said moieties are arranged in linear order (5' to 3') and linked to each other by a nucleosidic bond, thereby forming a modular nucleic acid molecule.
  • a polypeptide or protein molecule comprises multiple (two or more) elements, said elements are arranged in linear order (from N-terminus to C-terminus), and linked to each other by a peptide bond, thereby forming a modular polypeptide or protein.
  • Such modular polypeptide or protein comprises multiple elements in linear order, with respect to the polypeptide strand.
  • the coding region can be designed or tailored on nucleic acid level.
  • a tailored or designed nucleic acid molecule e.g. RNA, e.g. mRNA, consists of several moieties, each consisting of a nucleic acid sub-sequence.
  • the tai lored or designed nucleic acid molecule comprises (i) at least one nucleic acid moiety encoding at least one polypeptide of interest (e.g. a protein potentially producing a therapeutic outcome) and (ii) preferably at least one further nucleic acid moiety.
  • Said further nucleic acid moiety may be selected among coding moieties and non-coding moieties. More than one of such moieties can be present in an optimized nucleic acid molecule.
  • the optimized nucleic acid molecule of the present invention is characterized by addition of at least one nucleic acid moiety (coding or non-coding), as described herein. Said addition is preferably realized 5' or 3' with respect to a starting (e.g. wi ld-type) nucleic acid molecule.
  • said coding moiety encodes for an element conferring a feature that is beneficial in the context of the polypeptide of interest, e.g. for an envisaged therapeutic application.
  • Such further elements may be selected among a secretory signal peptide (SSP), a multimerization element, a virus like particle (VLP) forming element, a transmembrane element, a dendritic cell targeting element, an immunological adjuvant element, an element promoting antigen presentation; a 2A peptide; a peptide linker element, an element directing post-translational modification (e.g. glycosylation), and/or any other polypeptide or protein.
  • Further non-coding moieties are selected from the group comprising 3'-UTR, 5'-UTR, IRES, miRNA binding site, histone stem loop, poly(A)-sequence and/or any other polynucleotide moiety.
  • the polypeptide or protein element of interest may for example be selected from the group comprising therapeutic proteins, therapeutic polypeptides, allergens, autoimmune antigens, pathogenic antigens, and tumour antigens.
  • the optimized nucleic acid molecule is (also) characterized by the presence of at least one chemical modification, e.g. at least one modified nucleoside.
  • at least one nucleoside deoxyribonucleoside or ribonucleoside
  • the chemical modification is a structural feature of such optimized nucleic acid molecule.
  • the at least one distinguishing structural feature provides - or contributes to - a functional property of the optimized nucleic acid molecule which is not exhibited by the non- optimized (wild-type) nucleic acid molecule.
  • functional properties can be selected from the list comprising improved or increased RNA stability, improved or directed RNA localization, improved or increased RNA lifetime, improved or increased translation of the RNA, improved or increased stability of the encoded polypeptide or protein, tissue- or target cell-specific expression of the encoded polypeptide or protein, improved or target-compartment directed localization of the encoded polypeptide or protein, such as localization at a membrane or in soluble form, in a particular cell organelle, at the cell surface, in excreted form, and the like.
  • Functional properties also include properties associated with multimerization or particle formation of the polypeptide. Further, functional properties may i nclude an added function, such as mediated by a fusion protein, wherein the added function is provided by a second polypeptide element. For such purposes, the nucleic acid moiety encoding second polypeptide is fused in frame with respect to the polypeptide of interest. Any two or more such functional properties may be exhibited by an optimized nucleic acid molecule of the present invention. 1 . Type of nucleic acid molecule of the present invention
  • the optimized nucleic acid molecule according to the present invention may be RNA, such as mRNA or viral RNA or a replicon, DNA, such as a DNA plasmid or viral DNA, or may be a modified RNA or DNA molecule. It may be provided as a double-stranded molecule having a sense strand and an anti-sense strand, for example, as a DNA molecule having a sense strand and an anti-sense strand.
  • the invention provides an optimized nucleic acid molecule which is a DNA molecule.
  • Such nucleic acid molecule may serve as a template for an RNA molecule, preferably for an mRNA molecule.
  • the optimized nucleic acid molecule may be a DNA which may be used as a template for production of an RNA e.g. an mRNA or a replicon.
  • An mRNA is preferable.
  • the obtainable RNA may, in turn, be translated for production of a desired peptide or protein encoded by the open reading frame.
  • the optimized nucleic acid molecule is a DNA, it may, for example, be used as a double-stranded storage form for continued and repetitive in vitro or in vivo production of RNA e.g. mRNA.
  • RNA is the preferred nucleic acid molecule.
  • the nucleic acid molecule of the invention is an RNA molecule.
  • RNA molecules may be obtainable by transcription from a DNA molecule according to the present invention.
  • RNA molecules may also be obtainable in vitro by common methods of chemical synthesis, without being necessarily transcribed from a DNA progenitor.
  • RNA has numerous advantages over DNA as the nucleic acid for a genetic vehicle, including: i) The RNA introduced into the cell does not integrate into the genome (whereas DNA does integrate into the genome to a certain degree and can also be inserted into an intact gene of the genome of the host cell, causing a mutation of the respective gene, which can lead to a partial or total loss of the genetic information or to misinformation).
  • RNA No viral sequences, such as promoters etc., are required for the effective transcription of RNA (whereas a strong promoter (e.g. the viral CMV promoter) is required for the expression of DNA introduced into the cell).
  • a strong promoter e.g. the viral CMV promoter
  • the integration of such promoters into the genome of the host cell can lead to undesirable changes in the regulation of gene expression.
  • the degradation of RNA that has been introduced takes place in a limited period of time, so that it is possible to achieve transient gene expression, which can be discontinued after the required treatment period (whereas this is not possible in the case of DNA that has been integrated into the genome).
  • RNA does not lead to the induction of pathogenic anti-RNA antibodies in the patient (whereas the induction of anti-DNA antibodies is known to cause an undesirable immune response).
  • RNA is widely applicable; any desired RNA for any desired protein of interest can be prepared in short period of time for therapeutic purposes, even on an individual patient basis (personalized medicine).
  • the RNA molecule preferably comprises at least one further coding or non-coding moiety, such as an untranslated region (UTR).
  • UTR untranslated region
  • the invention provides an optimized RNA molecule, preferably an artificial mRNA molecule or an artificial viral RNA molecule.
  • the RNA of the present invention is messenger RNA (mRNA), i.e. RNA encoding at least one polypeptide or protein.
  • mRNA species corresponds to a genomic transcription unit.
  • the optimized nucleic acid molecule can be designed by combination of more than one nucleic acid moieties.
  • the term “moiety” or “nucleic acid moiety” is used herein to refer to a unit, or building block, on nucleic acid level
  • the term “element” or “polypeptide element” or “polypeptide or protein element” is used herein to refer to a unit, or building block, on polypeptide or protein level.
  • the present invention allows to incorporate and recombine desired coding and non-coding moieties into a single nucleic acid molecule.
  • At least one such moiety is a coding moiety, i.e. a nucleic acid moiety encoding a polypeptide element or protein element. Whether any particular moiety is desired or not depends on the circumstances. For example, if multimerization of an encoded polypeptide is intended, a multimerization element is a desired element of the encoded polypeptide, and thus the nucleic acid sequence coding therefor is a desired moiety of an optimized nucleic acid encoding such polypeptide capable of multimerization, and so on.
  • nucleic acid moieties e.g. encoding polypeptide or protein elements
  • suitable nucleic acid moieties e.g. encoding polypeptide or protein elements
  • Suitable moieties can generally be selected from coding moieties and from non-coding moieties.
  • a nucleic acid molecule in the context of the present invention is typically the provision of genetic information encoding a polypeptide or protein, at least one coding moiety is typically comprised.
  • the polynucleotide of the present invention is preferably artificial or chimeric.
  • a chimeric molecule e.g. polynucleotide, comprises typically sequence information originating from more than one protein and/or from more than one species.
  • nucleic acid molecules comprising several moieties are known to the person skilled in the art and include, without limitation, in vitro synthesis and molecular biological approaches, e.g. enzymatic linkage of nucleic acid fragments by the help of ligase enzymes.
  • Coding moieties can be selected from nucleic acid sequences encoding one or more polypeptides from the following list:
  • nucleic acid sequence encoding a polypeptide or protein of interest
  • SSP secretory signal peptide
  • nucleic acid sequence encoding a multimerization element including dimerization, trimerization, tetramerization or oligomerization elements
  • VLP virus like particle
  • a nucleic acid sequence encoding a transmembrane element — a nucleic acid sequence encoding a dendritic cell targeting element
  • a polypeptide or protein of interest is any polypeptide or protein that is of interest for the desired purpose.
  • a polypeptide or protein of interest can be referred to herein as target protein/polypeptide
  • target protein/polypeptide when the purpose is vaccination against a certain antigen, a polypeptide or protein of interest is a polypeptide or protein which possesses the respective antigenic determinant.
  • the nucleic acid molecule of the present invention comprises at least one moiety encoding a polypeptide or protein of interest, and optionally additionally one or more further moiety encoding a further element from the above list.
  • the nucleic acid molecule encodes at least one additional polypeptide or protein element
  • the at least one additional polypeptide or protein element is encoded in the same reading frame as the polypeptide or protein of interest.
  • Proteins or polypeptides encoded by that type of nucleic acids are also referred to as fusion proteins.
  • the nucleic acid molecule of the present invention comprises a fusion protei n.
  • a fusion protein can comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more polypeptide elements or protein elements.
  • Non-coding moieties can be selected from nucleic acid sequences from one or more disclosed the following list:
  • At least one 5'-UTR moiety and/or at least one 3'-UTR moiety is selected.
  • at least one 5'-UTR and at least one 3'-UTR is selected.
  • More than one coding moiety can be comprised in the optimized nucleic acid molecule, such as 2 coding moieties, 3 coding moieties, 4 coding moieties, 5 coding moieties, 6 coding moieties, 7 coding moieties, 8 codi ng moieties, 9 coding moieties, 1 0 coding moieties or more than 1 0 coding moieties. It is also possible that 2 to 1 0 coding moieties, 3 to 9 coding moieties, four to eight coding moieties, five to seven coding moieties are comprised.
  • the coding moieties encode the respective polypeptide elements or protein elements in the same open reading frame, translation of said open reading frame will result in the expression of a fusion protein.
  • Such a fusion protein can comprise the respective number of polypeptide or protein elements.
  • More than one non-coding moiety can be comprised, such as 2 non-coding moieties, 3 non- coding moieties, 4 non-coding moieties, 5 non-coding moieties, 6 non-coding moieties, 7 non-coding moieties, 8 non-coding moieties, 9 non-coding moieties, 1 0 non-coding moieties or more than 1 0 non-coding moieties. It is also possible that two to ten non-coding moieties, three to nine non-coding moieties, four to eight non-coding moieties, five to seven non- coding moieties are comprised. Typically, at least a 3'-UTR moiety and/or at least a 5'-UTR moiety are comprised.
  • any combination of moieties (coding moieties and non- coding moieties) is possible.
  • the skilled person can routinely select appropriate moieties.
  • the present invention allows for the versati le combination and recombination of nucleic acid moieties, and thus of polypeptide/protein elements.
  • a nucleic acid which is fit for any given purpose can be designed and prepared.
  • an optimal nucleic acid for any given purpose can be designed and prepared. Since such optimal nucleic acid, or optimized nucleic acid, is provided by the present invention, the present invention concerns not only such nucleic acid as such, but also methods for their preparation and apparatuses for their preparation.
  • the present invention thus al lows for versati le recombination of nucleic acid moieties for any given purpose.
  • Versati lity is achieved by combination of moieties (coding moieties and non- coding moieties) as disclosed herein. 3.
  • moieties coding moieties and non- coding moieties
  • polypeptide or protein of interest is not limited. Rather, in line with the general concept of the present invention, virtually any polypeptide or protein, or nucleic acid encoding such polypeptide or protein, can be used or employed.
  • Non-limiting examples include proteins of human origin, proteins of animal origin, proteins of plant origin, proteins of protozoological origin, proteins of virus origin, proteins of bacterial or archaebacterial origin, chimeric proteins, artificial proteins.
  • the polypeptide or protein is encoded by an open reading frame (ORF).
  • the ORF does not encode a ribosomal protein of human or plant origin, in particular Arabidopsis origin, in particular does not encode human ribosomal protein S6 (RPS6), human ribosomal protein L36a-like (RPL36AL) or Arabidopsis ribosomal protein S1 6 (RPS1 6).
  • the open reading frame does not encode ribosomal protein S6 (RPS6), ribosomal protein L36a-like (RPL36AL) or ribosomal protein S1 6 (RPS1 6) of whatever origin.
  • the open reading frame of the optimized nucleic acid molecule according to the present invention does not code for a reporter protein, e.g., a reporter protein selected from the group consisting of globin proteins (particularly beta-globin), !uciferase protein, GFP proteins, glucuronidase proteins (particularly beta- glucuronidase) or variants thereof, for example, variants exhibiting at least 70% sequence identity to a globin protein, a luciferase protein, a GFP protein, or a glucuronidase protein.
  • a reporter protein selected from the group consisting of globin proteins (particularly beta-globin), !uciferase protein, GFP proteins, glucuronidase proteins (particularly beta- glucuronidase) or variants thereof, for example, variants exhibiting at least 70% sequence identity to a globin protein, a luciferase protein, a GFP protein, or a glucuronidase protein.
  • the at least one open reading frame encodes a therapeutic protein or peptide.
  • an antigen is encoded by the at least one open reading frame, such as a pathogenic antigen, a tumour antigen, an allergenic antigen or an autoimmune antigen.
  • an antibody or an antigen-specific T cell receptor or a fragment thereof is encoded by the at least one open reading frame of the optimized nucleic acid molecule according to the invention.
  • suitable polypeptides and proteins of interest include pathogenic antigens, tumour antigens, and therapeutic proteins. Such examples are described below.
  • the protein or polypeptide may comprise or consist of a therapeutic protein, a fragment, variant or derivative of a protein or a peptide, which comprises a therapeutic protein or a fragment, variant or derivative thereof.
  • Therapeutic proteins as defined herein are peptides or proteins, which are beneficial for the treatment of any inherited or acquired disease or which improves the condition of an individual. Particularly, therapeutic proteins play an important role in the creation of therapeutic agents that could modify and repair genetic errors, destroy cancer cells or pathogen infected cells, treat immune system disorders, treat metabolic or endocrine disorders, among other functions. For instance, Erythropoieti (EPO), a protein hormone can be utilized in treating patients with erythrocyte deficiency, which is a common cause of kidney complications. Furthermore adjuvant proteins, therapeutic antibodies are encompassed by therapeutic proteins and also hormone replacement therapy which is e.g. used in the therapy of women in menopause.
  • EPO Erythropoieti
  • therapeutic antibodies are encompassed by therapeutic proteins and also hormone replacement therapy which is e.g. used in the therapy of women in menopause.
  • somatic cells of a patient are used to reprogram them into pluripotent stem cells, which replace the disputed stem cell therapy.
  • these proteins used for reprogramming of somatic cells or used for differentiating of stem cells are defined herein as therapeutic proteins.
  • therapeutic proteins may be used for other purposes, e.g. wound healing, tissue regeneration, angiogenesis, etc.
  • antigen-specific B cell receptors and fragments and variants thereof are defined herein as therapeutic proteins.
  • therapeutic proteins can be used for various purposes including treatment of various diseases like e.g. infectious diseases, neoplasms (e.g. cancer or tumour diseases), diseases of the blood and blood-forming organs, endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system, independently if they are inherited or acquired.
  • infectious diseases e.g. infectious diseases, neoplasms (e.g. cancer or tumour diseases)
  • diseases of the blood and blood-forming organs e.g. cancer or tumour diseases
  • diseases of the blood and blood-forming organs e.g., endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue,
  • particularly preferred therapeutic proteins which can be used inter alia in the treatment of metabolic or endocrine disorders are selected from (in brackets the particular disease for which the therapeutic protein is used in the treatment): Acid sphingomyelinase (Niemann-Pick disease), Adipotide (obesity), Agalsidase-beta (human galactosidase A) (Fabry disease; prevents accumulation of lipids that could lead to renal and cardiovascular complications), Alglucosidase (Pompe disease (glycogen storage disease type II)), alpha- galactosidase A (alpha-GAL A, Agalsidase alpha) (Fabry disease), alpha-glucosidase (Glycogen storage disease (GSD), Morbus Pompe), alpha-L-iduronidase (mucopolysaccharidoses (MPS), Hurler syndrome, Scheie syndrome), alpha-N- acetylglucosaminidase (Sanfil
  • therapeutic proteins are understood to be therapeutic, as they are meant to treat the subject by replacing its defective endogenous production of a functional protein in sufficient amounts. Accordingly, such therapeutic proteins are typically mammalian, in particular human proteins.
  • tPA tissue plasminogen activator
  • infectious diseases or immunedeficiencies following therapeutic proteins may be used: Alteplase (tissue plasminogen activator; tPA) (Pulmonary embolism, myocardial infarction, acute ischaemic stroke, occlusion of central venous access devices), Anistreplase (Thrombolysis), Antithrombin 111 (AT-III) (Hereditary AT- III deficiency, Thromboembolism), Bivalirudin (Reduce blood-clotting risk in coronary angioplasty and heparin-induced thrombocytopaenia), Darbepoetin-alpha (Treatment of anaemia in patients with chronic renal insufficiency
  • the protein or a polypeptide of interest may consist or comprise of a pathogenic antigen or a fragment, variant or derivative thereof.
  • pathogenic antigens are derived from pathogenic organisms, in particular bacterial, viral or protozoological pathogenic organisms, which evoke an immunological reaction in a subject, in particular a mammalian subject, more particularly a human.
  • pathogenic antigens are preferably surface antigens, e.g. proteins (or fragments of proteins, e.g. the exterior portion of a surface antigen) located at the surface of the virus or the bacterial or protozoological organism.
  • Pathogenic antigens are peptide or protein antigens preferably derived from a pathogen associated with infectious disease which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophi lum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Bur
  • antigens from the pathogens selected from Influenza virus, respiratory syncytial virus (RSV), Herpes simplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiency virus (HIV), Plasmodium, Staphylococcus aureus, Dengue virus, Chlamydia trachomatis, Cytomegalovirus (CMV), Hepatitis B virus (HBV), Mycobacterium tuberculosis, Rabies virus, and Yellow Fever Virus.
  • RSV respiratory syncytial virus
  • HSV Herpes simplex virus
  • HPV human Papilloma virus
  • HIV Human immunodeficiency virus
  • Plasmodium Staphylococcus aureus
  • Dengue virus Chlamydia trachomatis
  • Cytomegalovirus CMV
  • HBV Hepatitis B virus
  • Mycobacterium tuberculosis Rabies virus
  • Yellow Fever Virus 3.1 .1 .
  • the protein or polypeptide may comprise or consist of a tumour antigen, a fragment, variant or derivative of a tumour antigen.
  • a tumour antigen is selected from the group comprising a melanocyte-specific antigen, a cancer-testis antigen or a tumour-specific antigen, preferably a CT-X antigen, a non-X CT-antigen, a binding partner for a CT-X antigen or a binding partner for a non-X CT-antigen or a tumour-specific antigen, more preferably a CT-X antigen, a binding partner for a non-X CT-antigen or a tumour-specific antigen or a fragment, variant or derivative of said tumour antigen; and wherein each of the nucleic acid sequences encodes a different peptide or protein; and wherein at least one of the nucleic acid sequences encodes for 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1
  • SSPs Secretory signal peptides
  • such signal sequence When used in combination with a polypeptide or protein of interest in the context of the present invention, such signal sequence is typically placed N-terminal to the polypeptide or protein of interest. On nucleic acid level, the coding sequence for such signal sequence is typically placed in frame (i.e. in the same reading frame), 5' to the coding sequence for the polypeptide or protein of interest.
  • Preferred secretory signal sequences are those functional in eukaryotic cells.
  • the secretory signal peptide is typically cleaved from the nascent polypeptide chain immediately after it has been translocated into the membrane of the endoplasmic reticulum.
  • the translocation occurs co-translationally and is dependent on a cytoplasmic protein-RNA complex (signal recognition particle, SRP).
  • SRP signal recognition particle
  • protein folding and certain post-translational modifications can occur (e.g., glycosylation). Then, the protein is typically transported into Golgi vesicles and eventually secreted. There is no well-defined consensus sequence or sequence motif for signal peptides, but there is a common structure.
  • Secretory signal sequences have a tripartite structure, consisting of a hydrophobic core region flanked by an n- and c-region. Typical ly, the n-region is one to five amino acids in length, carrying positively charged amino acids. Between the hydrophobic core region and the signal peptidase cleavage site is the c-region, which consists of three to seven polar, but mostly uncharged, amino acids. Close to the cleavage site a more specific pattern of amino acids, known as the (3, 1 )-rule, is found: the amino acid residues at positions 3 and 1 (relative to the cleavage site) must be small and neutral for cleavage to occur correctly.
  • a proper secretion of the antigen is beneficial for the induction of an immune response, because secretion of the antigen mimics the "natural" way of a viral infection and cytoplasmic localization of the expressed antigenic peptides/proteins could strongly limit the exposure of antigens to professional immune cells required for an induction of a humoral immune response.
  • Secretory signal peptides may be used as additional elements to promote or improve the secretion of the target protein (protein of interest).
  • the polypeptide sequence of the SSP used in the present invention is selected from the following list of polypeptide sequences (SEQ I D NOs: 1 -1 1 1 5 and SEQ ID NO: 1 728).
  • nucleic acid level particularly RNA level
  • any nucleotide sequence moiety can be employed that encodes any of SSP used in the present invention.
  • such nucleotide sequence is selected to encode a polypeptide selected from the following list of polypeptide sequences SEQ ID NOs: 1 -1 1 1 5 and SEQ ID NO: 1 728.
  • SEQ ID NOs: 1 -1 1 1 5 and SEQ ID NO: 1 728 Owing to the degenerated genetic code, in the case of most polypeptides SEQ ID NOs: 1 -1 1 1 5 and SEQ ID NO: 1 728, more than one particular nucleic acid sequence is conceivable as encoding the respective polypeptide. While each and every such nucleic acid may generally be used in the context of the present invention, it is preferable that the nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1 -1 1 15 and SEQ ID NO: 1 728).
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • multimerization of the encoded antigen may be beneficial for the induction of an immune response. Fusion of the target antigen to multimerization elements (e.g., dimerization elements, trimerization elements, tetramerization elements, and oiigomerization elements) may lead to the formation of multimeric antigen-complexes. This potentially increases immunogenicity of the respective antigen because such antigen-complexes may mimic a "natural" infection with an exogenous pathogen (e.g., virus) where a plurality of potential antigens is commonly located at the envelope of the pathogen (e.g., hemagglutinin (HA) antigen of the influenza virus).
  • pathogen e.g., virus
  • a plurality of potential antigens is commonly located at the envelope of the pathogen (e.g., hemagglutinin (HA) antigen of the influenza virus).
  • such multimerization element When used in combination with a polypeptide or protein of interest in the context of the present invention, such multimerization element can be placed N-terminal or C-terminal to the polypeptide of interest.
  • the coding sequence for such multimerization element On nucleic acid level, the coding sequence for such multimerization element is typically placed in frame (i.e. in the same reading frame), 5' or 3' to the coding sequence for the polypeptide or protein of interest.
  • Particular multimerization elements are oiigomerization elements, tetramerization elements, trimerization elements or dimerization elements.
  • Dimerization elements may be selected from e.g. dimerization elements/domains of heat shock proteins, immunoglobulin Fc domains and leucine zippers (dimerization domains of the basic region leucine zipper class of transcription factors). Specific elements are provided in SEQ ID NOs. 1 1 16 - 1 120. Trimerization and tetramerization elements may be selected from e.g.
  • engineered leucine zippers engineered a-helical coiled coil peptide that adopt a parallel trimeric state
  • fibritin foldon domain from enterobacteria phage T4, GCN4pll, CCN4-pLI, and p53.
  • Specific elements are provided in SEQ ID NOs. 1 121 -1 145 (trimerization elements) and SEQ ID NOs. 1 1 46-1 149 (tetramerization elements).
  • Oligomerization elements may be selected from e.g. ferritin, surfactant D, oligomerization domains of phosphoproteins of paramyxoviruses, complement inhibitor C4 binding protein (C4bp) oligomerization domains, Viral infectivity factor (Vif) oligomerization domain, sterile alpha motif (SAM) domain, and von Wil lebrand factor type D domain.
  • ferritin e.g. ferritin
  • surfactant D oligomerization domains of phosphoproteins of paramyxoviruses
  • C4bp complement inhibitor C4 binding protein
  • Vif Viral infectivity factor
  • SAM sterile alpha motif
  • von Wil lebrand factor type D domain e.g. ferritin, surfactant D, oligomerization domains of phosphoproteins of paramyxoviruses, complement inhibitor C4 binding protein (C4bp) oligomerization domains, Viral infectivity factor (Vif) oligomerization
  • Ferritin forms oligomers and is a highly conserved protein found in all animals, bacteria, and plants. Ferritin is a protein that spontaneously forms nanoparticles of 24 identical subunits. Ferritin-antigen fusion constructs potentially form oligomeric aggregates or "clusters" of antigens that may enhance the immune response.
  • SPD Surfactant D protein
  • An SPD-antigen fusion constructs may form oligomeric aggregates or "clusters" of antigens that may enhance the immune response.
  • Phosphoprotein of paramyxoviruses functions as a transcriptional transactivator of the viral polymerase. Oligomerization of the phosphoprotein is critical for viral genome replication. A phosphoprotein-antigen fusion constructs may form oligomeric aggregates or "clusters" of antigens that may enhance the immune response.
  • Complement inhibitor C4 binding Protein may also be used as a fusion partner to generate oligomeric antigen aggregates.
  • the C -terminal domain of C4bp (57 amino acid residues in humans and 54 amino acid residues in mice) is both necessary and sufficient for the oligomerization of C4bp or other polypeptides fused to it.
  • a C4bp-antigen fusion constructs may form oligomeric aggregates or "clusters" of antigens that may enhance the immune response.
  • Viral infectivity factor (Vif) multimerization domain has been shown to form oligomers both in vitro and in vivo.
  • Vif The oligomerization of Vif involves a sequence mapping between residues 1 51 to 1 64 in the C-terminal domain, the 1 61 PPLP1 64 motif (for human HIV-1 : TPKKIKPPLP).
  • a Vif-antigen fusion constructs may form oligomeric aggregates or "clusters" of antigens that may enhance the immune response.
  • the sterile alpha motif (SAM) domain is a protein interaction module present in a wide variety of proteins involved in many biological processes.
  • SAM domain that spreads over around 70 residues is found in diverse eukaryotic organisms.
  • SAM domains have been shown to homo- and hetero-oligomerise, forming multiple self-association oligomeric architectures.
  • a SAM-antigen fusion constructs may form oligomeric aggregates or "clusters" of antigens that may enhance the immune response.
  • von Willebrand factor (vWF) contains several type D domains: D1 and D2 are present within the N-terminal propeptide whereas the remaining D domains are required for oligomerization.
  • the vWF domain is found in various plasma proteins: complement factors B, C2, C 3 and CR4; the Integrins (l-domains); collagen types VI, VII, XII and XIV; and other extracellular proteins.
  • a vWF-antigen fusion constructs may form oligomeric aggregates or "clusters" of antigens that may enhance the immune response.
  • Multimerization elements useful in the present invention are provided in SEQ ID NOs: 1 1 1 6- 1 1 67. Multimerization elements fused to respective target proteins (antigens) may be used to form antigen nanoparticles.
  • polypeptide sequence of the multimerization element used in the present invention is selected from the following list of polypeptide sequences (SEQ ID NOs: 1 1 1 6- 1 1 67).
  • nucleic acid level particularly RNA level
  • any nucleotide sequence moiety can be employed that encodes any of oligomerization element used in the present invention.
  • such nucleotide sequence is selected to encode a polypeptide selected from the following list of polypeptide sequences SEQ ID NOs: 1 1 1 6-1 1 67.
  • SEQ ID NOs: 1 1 1 6-1 1 67 Owing to the degenerated genetic code, in the case of most polypeptides SEQ ID NOs: 1 1 1 6-1 1 67, more than one particular nucleic acid sequence is conceivable as encoding the respective polypeptide. While each and every such nucleic acid may generally be used in the context of the present invention, it is preferable that the nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1 1 1 6-1 1 67.
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • VLP Virus like particle
  • VLPs are self-assembled viral structural proteins (envelope proteins or capsid proteins) that structurally resemble viruses (without containing viral genetic material). VLPs contain repetitive high density displays of antigens which present conformational epitopes that can elicit strong T cell and B cell immune responses.
  • VLP forming element When used in combination with a polypeptide or protein of interest in the context of the present invention, such VLP forming element can be placed N-terminal or C-terminal to the polypeptide of interest.
  • the coding sequence for such VLP forming element is typically placed in frame (i .e. in the same reading frame), 5' or 3' to the coding sequence for the polypeptide or protein of interest.
  • VLP formi ng elements fused to an antigen may generate virus like particles containing repetitive high density displays of antigens.
  • VLP formi ng elements may be selected e.g. from any one of SEQ ID NOs: 1 1 68-1227. Essentially, such VLP forming elements can be chosen from any viral or phage capsid or envelope protein.
  • VLP forming elements may be used as additional elements to promote or improve the particle formation of the target protein.
  • the polypeptide sequence of the VLP forming element used in the present invention is selected from the following list of polypeptide sequences (SEQ ID NOs: 1 1 68-1227).
  • nucleic acid level particularly RNA level
  • any nucleotide sequence moiety can be employed that encodes any of VLP forming element used in the present invention.
  • such nucleotide sequence is selected to encode a polypeptide selected from the following list of polypeptide sequences SEQ ID NOs: 1 1 68-1227. Owing to the degenerated genetic code, in the case of most polypeptides SEQ ID NOs: 1 168-1227, more than one particular nucleic acid sequence is conceivable as encoding the respective polypeptide of the below list. While each and every such nucleic acid may generally be used in the context of the present invention, it is preferable that the nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1 1 68-1227.
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • Transmembrane elements or membrane spanning polypeptide elements are present in proteins that are integrated or anchored in plasma membranes of cells.
  • Typical transmembrane elements are alpha-helical transmembrane elements.
  • Such transmembrane elements are composed essentially of amino acids with hydrophobic side chains, because the interior of a cell membrane (lipid bilayer) is also hydrophobic.
  • transmembrane elements are commonly single hydrophobic alpha helices or beta barrel structures; whereas hydrophobic alpha helices are usually present in proteins that are present in membrane anchored proteins (e.g., seven transmembrane domain receptors), beta-barrel structures are often present in proteins that generate pores or channels.
  • target proteins such as antigens associated with infectious (e.g.
  • transmembrane element may be beneficial to introduce a transmembrane element into the respective constructs.
  • a transmembrane element to the target peptide/protein it may be possible to further enhance the immune response, wherein the translated target peptide/protein, e.g. a viral antigen, anchors to a target membrane, e.g. the plasma membrane of a cell, thereby increasing immune responses. This effect is also referred to as antigen clustering.
  • transmembrane element When used in combination with a polypeptide or protein of interest in the context of the present invention, such transmembrane element can be placed N-terminal or C-terminal to the polypeptide of interest.
  • the coding sequence for such transmembrane element is typically placed in frame (i.e. in the same reading frame), 5' or 3' to the coding sequence for the polypeptide or protein of interest.
  • the transmembrane domain may be selected from the transmembrane domain of Hemagglutinin (HA) of Influenza virus, Env of HIV-1 , EIAV (equine infectious anaemia virus), MLV (murine leukaemia virus), mouse mammary tumor virus, G protein of VSV (vesicular stomatitis virus), Rabies virus, or a transmembrane element of a seven transmembrane domain receptor. Specific elements are provided in the Table below.
  • TM transmembrane
  • the polypeptide sequence of the transmembrane (TM) domain used in the present invention is selected from the following list of polypeptide sequences (SEQ ID NOs: 1228- 1343).
  • any nucleotide sequence moiety can be employed that encodes any transmembrane (TM) domain used in the present invention.
  • such nucleotide sequence is selected to encode a polypeptide selected from the following list of polypeptide sequences SEQ ID NOs: 1228-1 343.
  • SEQ ID NOs: 1228-1343 Owing to the degenerated genetic code, in the case of most polypeptides SEQ ID NOs: 1228-1343, more than one particular nucleic acid sequence is conceivable as encoding the respective polypeptide . While each and every such nucleic acid may generally be used in the context of the present invention, it is preferable that the nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1228-1343.
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • Dendritic cells the most potent antigen presenting cells (APCs), link the innate immune response to the adaptive immune response. They bind and internalize pathogens/antigens and display fragments of the antigen on their membrane (via MHC molecules) to stimulate T-cell responses against those pathogens/antigens.
  • Polypeptide elements capable of targeting to dendritic cells are referred to as dendritic cell targeting elements.
  • such dendritic cell targeting element When used in combination with a polypeptide or protein of interest in the context of the present invention, such dendritic cell targeting element can be placed N-terminal or C- terminal to the polypeptide of interest.
  • the coding sequence for such dendritic cell element On nucleic acid level, is typically placed in frame (i.e. in the same reading frame), 5' or 3' to the coding sequence for the polypeptide or protein of interest.
  • Targeting antigens to DCs is an appropriate method to stimulate and induce effective antitumor and antiviral immune responses.
  • proteins/peptides e.g., antibody fragments, receptor ligands
  • bind to DC surface receptors have to be fused to the respective antigen/target protein.
  • Such DC receptors include C-type lectins (mannose receptors (e.g., MR1 , DEC-205 (CD205)), CD206, DC-SIGN (CD209), Clec9a, DCIR, Lox-1 , MGL, MGL-2, Clec12A, Dectin-1 , Dectin-2, langerin (CD207)), scavenger receptors, F4/80 receptors (EMR1 ), DC-STAMP, receptors for the Fc portion of antibodies (Fc receptors), toll-like receptors (e.g., TLR2, 5, 7, 8, 9) and complement receptors (e.g., CR1 , CR2).
  • C-type lectins mannose receptors (e.g., MR1 , DEC-205 (CD205)), CD206, DC-SIGN (CD209), Clec9a, DCIR, Lox-1 , MGL, MGL-2, Clec12A, Dectin-1 , Dect
  • An antigen may be fused to the following elements to obtain targeting of dendritic cells: anti- DC-SIGN antibody, CD1 1 c specific single chain fragments (scFV), DEC205-specific single chain fragments (scFV), soluble PD-1 , chemokine (C motif) ligand XCL1 , CD40 ligand, human IgGI , murine lgG2a, anti Celec 9A, anti MHCII scFV.
  • anti- DC-SIGN antibody CD1 1 c specific single chain fragments (scFV), DEC205-specific single chain fragments (scFV), soluble PD-1 , chemokine (C motif) ligand XCL1 , CD40 ligand, human IgGI , murine lgG2a, anti Celec 9A, anti MHCII scFV.
  • any other protein/peptide element that binds to a receptor localized on dendritic cel ls may be used as an element (Apostolopoulos, Vasso, et al. "Targeting antigens to dendritic cell receptors for vaccine development.” Journal of drug delivery 201 3 (201 3); KastenmCiller, Wolfgang, et al. "Dendritic cell-targeted vaccines - hope or hype?” Nature Reviews Immunology 14.1 0 (2014): 705-71 1 ). Such dendritic cell antigens are also contemplated in the present i nvention.
  • the polypeptide sequence of the dendritic cell targeting element used in the present invention is selected from the following list of polypeptide sequences (SEQ ID NOs: 1 344- 1 359).
  • any nucleotide sequence moiety can be employed that encodes any of dendritic cel l targeting elements used in the present invention.
  • such nucleotide sequence is selected to encode a polypeptide selected from the following list of polypeptide sequences SEQ ID NOs: 1 344-1359. Owing to the degenerated genetic code, in the case of most polypeptides SEQ ID NOs: 1 344-1 359, more than one particular nucleic acid sequence is conceivable as encoding the respective polypeptide.
  • nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1 344-1 359.
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • Immunologic adjuvant elements may comprise peptide or protein elements that potentiate or "govern" the immune response.
  • Such elements may include peptides/proteins that trigger a danger response (e.g., damage-associated molecular pattern molecules (DAMPs)), elements that activate the complement system (e.g., peptides/proteins involved in the classical complement pathway, the alternative complement pathway, and the lectin pathway) or elements that activate an innate immune response (e.g., pathogen- associated molecular pattern molecules, PAMPs).
  • DAMPs damage-associated molecular pattern molecules
  • PAMPs pathogen- associated molecular pattern molecules
  • such immunologic adjuvant element When used in combination with a polypeptide or protein of interest in the context of the present invention, such immunologic adjuvant element can be placed N-terminal or C- terminal to the polypeptide of interest.
  • the coding sequence for such immunologic adjuvant element On nucleic acid level, is typically placed in frame (i.e. in the same reading frame), 5' or 3' to the coding sequence for the polypeptide or protein of interest.
  • target peptides/proteins such as antigens associated with infectious diseases or antigens associated with tumor diseases it may be beneficial to fuse the respective target peptide/protein to elements that potentiate the immune response against the target peptide/protein or shunts the immune response against the target peptide/protein towards a desired response (e.g., humoral or cellular response).
  • Immunologic adjuvant elements that may be fused to a target protein, may be selected from heat shock proteins (e.g., HSP60, HSP70, gp96), flagellin FliC, high mobility group box 1 proteins (e.g., HMGN1 ), extra domain A of fibronectin (EDA), C3 protein fragments (e.g.
  • polypeptide sequences are provided in the table below (polypeptide sequences).
  • the polypeptide sequence of the adjuvant element used in the present invention is selected from the following list of polypeptide sequences (SEQ ID NOs: 1360-1421 ).
  • SEQ ID NOs: 1360-1421 On nucleic acid level, particularly RNA level, any nucleotide sequence moiety can be employed that encodes any of adjuvant element used in the present invention.
  • such nucleotide sequence is selected to encode a polypeptide selected from the following list of polypeptide sequences SEQ ID NOs: 1 360-1421 . Owing to the degenerated genetic code, in the case of most polypeptides SEQ ID NOs: 1360-1 421 , more than one particular nucleic acid sequence is conceivable as encoding the respective polypeptide. While each and every such nucleic acid may generally be used in the context of the present invention, it is preferable that the nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1 360-1421 .
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • APCs antigen-presenting cel ls
  • MHC major histocompatibility complex
  • target peptides/proteins such as antigens associated with infectious diseases or antigens associated with tumor diseases it may be beneficial to fuse the respective target peptide/protein to elements that promote antigen presentation.
  • elements may comprise peptides/proteins that trigger the entry into the lysosome/proteasome pathway or that promote the entry into the exosome.
  • immunologic adjuvant element can be placed N-terminal or C-terminal to the polypeptide of interest.
  • some particular elements may be particularly functional when they are present either at the N-terminus, or at the C-terminus, i.e.
  • the coding sequence for such immunologic adjuvant element is typically placed in frame (i.e. in the same reading frame), 5' or 3' (in analogy to the respective wild-type context) to the coding sequence for the polypeptide or protein of interest.
  • Such elements promoting antigen presentation may be selected e.g. from MHC invariant chain (li), invariant chain (li) lysosome targeting signal, sorting signal of the lysosomal- associated membrane protein LAMP-1 , lysosomal integral membrane protein-ll (L!MP-ll), CI C2 Lactadherin domain.
  • MHC invariant chain invariant chain
  • li invariant chain
  • lysosome targeting signal sorting signal of the lysosomal- associated membrane protein LAMP-1
  • L!MP-ll lysosomal integral membrane protein-ll
  • CI C2 Lactadherin domain Specific elements are provided in the table below.
  • Elements promoting antigen presentation may be used as additional elements to promote or improve the secretion of the target protein.
  • the polypeptide sequence of the antigen-presentation promoting element used in the present invention is selected from the following list of polypeptide sequences (SEQ ID NOs: 1422-1433).
  • nucleic acid level particularly RNA level
  • any nucleotide sequence moiety can be employed that encodes any of the antigen-presentation promoting elements used in the present invention.
  • such nucleotide sequence is selected to encode a polypeptide selected from the following list of polypeptide sequences SEQ ID NOs: 1422- 1433. Owing to the degenerated genetic code, in the case of most polypeptides SEQ ID NOs: 1422-1433, more than one particular nucleic acid sequence is conceivable as encoding the respective polypeptide. While each and every such nucleic acid may generally be used in the context of the present invention, it is preferable that the nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1422-1433.
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element. 3.1 .9 2 A peptides
  • Viral 2A peptides (“self-cleaving" peptides) allow the expression of multiple proteins from a single open reading frame.
  • the terms 2A peptide and 2A element are used interchangeably herein.
  • the mechanism by the 2A sequence for generating two proteins from one transcript is by ribosome skipping - a normal peptide bond is impaired at 2 A, resulting in two discontinuous protein fragments from one translation event.
  • such 2A peptides are particularly useful when encoded by a nucleic acid encoding at least two functional protein elements.
  • a 2A element is useful when the nucleic acid molecule encodes at least one polypeptide or protein of interest and at least one further protein element.
  • a 2A element is present when the polynucleotide of the invention encodes two proteins or polypeptides of interest, e.g. two antigens.
  • the coding sequence for such 2A peptide is typically located in between the coding sequence of the polypeptide of interest and the coding sequence of the least one further protein element (which may also be a polypeptide of interest), so that cleavage of the 2A peptide leads to two separate polypeptide molecules, at least one of them being a polypeptide or protei n of interest.
  • target proteins that are composed of several polypeptide chai ns, such as antibodies
  • 2A peptides may also be beneficial when cleavage of the protein of interest from another encoded polypeptide element is desired.
  • 2A peptides may be derived from foot-and-mouth diseases virus, from equine rhinitis A virus, Thosea asigna virus, Porcine teschovirus-1 . Specific elements are provided in the table below.
  • the polypeptide sequence of the 2A peptide used in the present invention is selected from the following list of polypeptide sequences (SEQ ID NOs: 1 434-1 508).
  • SEQ ID NOs: 1 434-1 508 any nucleotide sequence moiety can be employed that encodes any of 2A peptide used in the present invention.
  • such nucleotide sequence is selected to encode a polypeptide selected from the following list of polypeptide sequences SEQ ID NOs: 1434-1 508.
  • nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1434-1 508.
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • protein constructs composed of several elements (e.g., target protein fused to a transmembrane domain)
  • the protein elements are often separated by peptide linker elements.
  • peptide linker elements The same applies for polypeptides of interest having various domains.
  • Such elements may be beneficial because they allow for a proper folding of the individual elements and thereby the proper functionality of each element.
  • spacer or "peptide spacer” is used herein.
  • linkers or spacers are particularly useful when encoded by a nucleic acid encoding at least two functional protein elements, such as at least one polypeptide or protein of interest and at least one further protein or polypeptide element, preferably also selected from the list of coding moieties of the present invention.
  • the linker is typically located on the polypeptide chain in between the polypeptide of interest and the at least one further protein element.
  • the coding sequence for such linker is typically placed in the reading frame, 5' or 3' to the coding sequence for the polypeptide or protein of interest, or placed between coding regions for individual polypeptide domains of a given protein of interest.
  • Peptide linkers are preferably composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids.
  • the small size of these amino acids provides flexibi lity, and allows for mobility of the connecting functional domains, as described by Chen et al. (Adv Drug Deliv Reb. 201 3; 65(10): 1 357-1 369).
  • the incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces an interaction between the linker and the protein moieties.
  • Rigid linkers generally maintain the distance between the protein domains and they may be based on helical structures and/or they have a sequence that is rich in proline.
  • Cleavable linkers (also termed “cleavage l inkers”) allow for in vivo separation of the protein domains.
  • the mechanism of cleavage may be based e.g. on reduction of disulfide bonds within the linker sequence or proteolytic cleavage.
  • the cleavage may be mediated by an enzyme (enzymatic cleavage), e.g. the cleavage linker may provide a protease sensitive sequence (e.g., furin cleavage).
  • a typical sequence of a flexible linker is composed of repeats of the amino acids Glycine (G) and Serine (S).
  • the linker may have the following sequence: GS, GSG, SGG, SG, GGS, SGS, GSS, SSG.
  • the same sequence is repeated multiple times (e.g. two, three, four, five or six times) to create a longer linker.
  • a single amino acid residue such as S or G can be used as a linker.
  • Peptide linkers including cleavage linkers, flexible linkers and rigid linkers, or spacers, may be selected from the ones shown in the table below.
  • Linkers or spacers may be used as additional elements to promote or improve the secretion of the target protein.
  • the polypeptide sequence of the linker or spacer used in the present invention is selected from the following list of polypeptide sequences (SEQ ID NOs: 1 509-1 565).
  • nucleic acid level particularly RNA level
  • any nucleotide sequence moiety can be employed that encodes any of linker or spacer used in the present invention.
  • nucleotide sequence is selected to encode a polypeptide selected from the fol lowing list of polypeptide sequences SEQ ID NOs: 1 509-1 565. Owing to the degenerated genetic code, in the case of most polypeptides of SEQ ID NOs: 1 509-1 565, more than one particular nucleic acid sequence is conceivable as encoding the respective polypeptide list.
  • nucleic acid sequence that encodes the polypeptide sequence is selected such that its sequence is codon-optimized according to the general guidance provided in this specification.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1 509-1 565.
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • protein elements described herein may be incorporated. Such half-life extending elements are particularly useful for target proteins that are smal ler than the kidney fi ltration cutoff of around 70 kDa and/or are subject to metabolic turnover by peptidases, which significantly limits their plasma half-life in vivo.
  • Elements that extend protein half-life may be derived from homo-amino acid polymer (HAPylation), albumin, the Fc portion of immmunoglobulins, albumin binding domains, albumin binding peptide, poly-glycine elements, elastin-like elements, transferrin, proline- alanine-serine polymers (PASylation), HCG beta-subunit CTP elements, XTEN derived elements, ELP elements (ELPylation), gelatin-like protein polymers, IgGI , lgG2, Ig binding domain of Staphylococcus, etc. Specific elements, without limiting the scope of the present invention, are provided in SEQ ID NOs: 1 671 - 1 727.
  • any other element that extends the half-life of the respective target protein may be suitable in the context of the present invention.
  • any polypeptide element may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the sequences SEQ ID NOs: 1 671 - 1 727.
  • any polynucleotide (e.g. RNA) moiety may be selected which encodes such polypeptide element.
  • the protein of interest may be fused or may comprise additional coding modules as listed below:
  • Elements suitable for targeting intracellular or extracel lular proteins including but not limited to cellular receptors
  • Element suitable for targeting cell surface molecules including but not limited to glycans and cel lular matrix components
  • At least one non-coding nucleic acid moiety is present in the optimized nucleic acid molecule of the present invention.
  • Untranslated regions are non-coding moieties of a nucleic acid sequence, particularly of an RNA sequence, preferably mRNA, sequence.
  • at least one untranslated region moiety is present in an RNA according to the present invention.
  • Suitable UTR moieties are selected from 5'-UTR moieties and 3'-UTR moieties.
  • the optimized nucleic acid according to the present invention comprises at least one open reading frame, at least one 3'-UTR (moiety) and at least one 5'-UTR (moiety).
  • the at least one 3'-UTR moiety and/or the at least one 5'-UTR moiety in the optimized nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence which is derived from the 3'-UTR and/or the 5'-UTR of a eukaryotic protein coding gene, preferably from the 3'-UTR and/or the 5'-UTR of a vertebrate protein coding gene, more preferably from the 3'-UTR and/or the 5'-UTR of a mammalian protein coding gene, e.g.
  • mouse and human protein coding genes even more preferably from the 3'-UTR and/or the 5'-UTR of a primate or rodent protein coding gene, in particular the 3'- UTR and/or the 5'-UTR of a human or murine protein coding gene.
  • the at least one 3'-UTR moiety in the optimized nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence which is preferably derived from a naturally (in nature) occurring 3'-UTR
  • the at least one 5'-UTR moiety in the optimized nucleic acid molecule according to the present invention comprises or consists of a nucleic acid sequence which is preferably derived from a naturally (in nature) occurring 5'-UTR.
  • the at least one open reading frame is heterologous to the at least one 3'-UTR moiety and/or to the at least one 5'-UTR moiety.
  • heterologous in this context means that two sequence moieties comprised by the optimized nucleic acid molecule, such as the open reading frame and the 3'-UTR moiety and/or the open reading frame and the 5'- UTR moiety, do not occur naturally (in nature) in this combination. They are typically recombinant.
  • the 3'-UTR moiety and/or the 5'-UTR moiety are/is derived from a different gene than the open reading frame.
  • the ORF may be derived from a different gene than the 3'-UTR moiety and/or to the at least one 5'-UTR moiety, e.g. encoding a different protein or the same protein but of a different species etc.
  • the open reading frame is derived from a gene which is distinct from the gene from which the 3'-UTR moiety and/or to the at least one 5'-UTR moiety is derived.
  • the ORF does not encode a human or plant (e.g., Arabidopsis) ribosomal protein, preferably does not encode human ribosomal protein S6 (RPS6), human ribosomal protein L36a-like (RPL36AL) or Arabidopsis ribosomal protein S1 6 (RPS16).
  • the open reading frame does not encode ribosomal protein S6 (RPS6), ribosomal protein L36a- like (RPL36AL) or ribosomal protein SI 6 (RPS1 6).
  • the open reading frame does not code for a reporter protein, e.g., selected from the group consisting of globin proteins (particularly beta- globin), luciferase protein, GFP proteins or variants thereof, for example, variants exhibiting at least 70% sequence identity to a globin protein, a luciferase protein, or a GFP protein.
  • a reporter protein e.g., selected from the group consisting of globin proteins (particularly beta- globin), luciferase protein, GFP proteins or variants thereof, for example, variants exhibiting at least 70% sequence identity to a globin protein, a luciferase protein, or a GFP protein.
  • the open reading frame does not code for a GFP protein.
  • the open reading frame does not encode a reporter gene or is not derived from a reporter gene, wherein the reporter gene is preferably not selected from group consisting of globin proteins (particularly beta-globin), luciferase protein, beta-glucuronidase (GUS) and GFP proteins or variants thereof, preferably not selected from EGFP, or variants of any of the above genes, typically exhibiting at least 70% sequence identity to any of these reporter genes, preferably a globin protein, a luciferase protein, or a GFP protein.
  • the reporter gene is preferably not selected from group consisting of globin proteins (particularly beta-globin), luciferase protein, beta-glucuronidase (GUS) and GFP proteins or variants thereof, preferably not selected from EGFP, or variants of any of the above genes, typically exhibiting at least 70% sequence identity to any of these reporter genes, preferably a globin protein, a luciferase protein, or a GFP protein.
  • the 3'-UTR moiety and/or the 5'-UTR moiety is heterologous to any other moiety comprised in the optimized nucleic acid as defined herein.
  • the optimized nucleic acid according to the invention comprises a 3'-UTR moiety from a given gene, it does preferably not comprise any other nucleic acid sequence, in particular no functional nucleic acid sequence (e.g. coding or regulatory sequence moiety) from the same gene, including its regulatory sequences at the 5' and 3' terminus of the gene's ORF.
  • the optimized nucleic acid according to the invention comprises a 5'-UTR moiety from a given gene, it does preferably not comprise any other nucleic acid sequence, in particular no functional nucleic acid sequence (e.g. coding or regulatory sequence moiety) from the same gene, including its regulatory sequences at the 5' and 3' terminus of the gene's ORF.
  • the at least one 3'-UTR moiety and/or the at least one 5'-UTR moiety is functionally linked to an open reading frame (ORF) of the optimized nucleic acid molecule.
  • ORF open reading frame
  • the 3'-UTR moiety and/or to the at least one 5'-UTR moiety is associated with the ORF such that it may exert a function, such as an enhancing or stabilizing function on the expression of the encoded peptide or protein or a stabilizing function on the optimized nucleic acid molecule.
  • the ORF and the 3'-UTR moiety are associated in 5'- 3' direction and/or the 5'-UTR moiety and the ORF are associated in 5'- 3' direction.
  • the optimized nucleic acid molecule comprises in general the structure 5'- [5'-UTR moiety]-(optional)-linker-ORF-(optional)-linker-[3'-UTR moiety]-3', wherein the optimized nucleic acid molecule may comprise only a 5'-UTR moiety and no 3'-UTR moiety, only a 3'-UTR moiety and no 5'-UTR moiety, or both, a 3'-UTR moiety and a 5'-UTR moiety.
  • the linker may be present or absent.
  • the linker may be one or more nucleotides, such as a stretch of 1 -50 or 1 -20 nucleotides, e.g., comprising or consisting of one or more restriction enzyme recognition sites (restriction sites).
  • the at least one 3'-UTR moiety and/or the at least one 5'-UTR moiety comprises or consists of a nucleic acid sequence which is derived from the 3'-UTR and/or the 5'-UTR of a transcript of a gene.
  • the at least one 3'-UTR moiety and/or the at least one 5'- UTR moiety of the optimized nucleic acid molecule according to the present invention comprises or consists of a "functional fragment", a "functional variant” or a "functional fragment of a variant" of the 3'-UTR and/or the 5'-UTR of a transcript of a gene.
  • nucleic acid sequence which is derived from the 3'-UTR and/or the 5'-UTR of a of a transcript of a gene preferably refers to a nucleic acid sequence which is based on the 3'-UTR sequence and/or on the 5'-UTR sequence of a transcript of a gene or a fragment or part thereof, preferably a naturally occurring gene or a fragment or part thereof.
  • the term naturally occurring is used synonymously with the term wild-type.
  • This phrase includes sequences corresponding to the entire 3'-UTR sequence and/or the entire 5'-UTR sequence, i.e.
  • a fragment of a 3'-UTR and/or a 5'-UTR of a transcript of a gene consists of a continuous stretch of nucleotides corresponding to a continuous stretch of nucleotides in the full-length 3'-UTR and/or 5'-UTR of a transcript of a gene, which represents at least 5%, 10%, 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, even more preferably at least 80%, and most preferably at least 90% of the full- length 3'-UTR and/or 5'-UTR of a transcript of a gene.
  • Such a fragment in the sense of the present invention, is preferably a functional fragment as described herein.
  • the fragment retains a regulatory function for the translation of the ORF linked to the 3'-UTR and/or 5'-UTR or fragment thereof.
  • variant of the 3'-UTR and/or variant of the 5'-UTR of a of a transcript of a gene refers to a variant of the 3'-UTR and/or 5'-UTR of a transcript of a naturally occurring gene, preferably to a variant of the 3'-UTR and/or 5'-UTR of a transcript of a vertebrate gene, more preferably to a variant of the 3'-UTR and/or 5'-UTR of a transcript of a mammalian gene, even more preferably to a variant of the 3'-UTR and/or 5'-UTR of a transcript of a primate gene, in particular a human gene as described above.
  • Such variant may be a modified 3'-UTR and/or 5'-UTR of a transcript of a gene.
  • a variant 3'-UTR and/or a variant of the 5'-UTR may exhibit one or more nucleotide deletions, insertions, additions and/or substitutions compared to the naturally occurring 3'-UTR and/or 5'-UTR from which the variant is derived.
  • a variant of a 3'-UTR and/or variant of the 5'-UTR of a of a transcript of a gene is at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% identical to the naturally occurring 3'-UTR and/or 5'-UTR the variant is derived from.
  • the variant is a functional variant as described herein.
  • the terms “functional variant”, “functional fragment”, and “functional fragment of a variant” in the context of the present invention, mean that the fragment of the 3'-UTR and/or the 5'-UTR, the variant of the 3'-UTR and/or the 5'-UTR, or the fragment of a variant of the 3'-UTR and/or the 5'-UTR of a transcript of a gene fulfils at least one, preferably more than one function of the naturally occurring 3'-UTR and/or 5'-UTR of a transcript of a gene of which the variant, the fragment, or the fragment of a variant is derived.
  • Such function may be, for example, stabilizing mRNA and/or enhancing, stabi lizing and/or prolonging protein production from an mRNA and/or increasing protein expression or total protein production from an mRNA, preferably in a mammalian cell, such as in a human cell.
  • the function of the 3'-UTR and/or the 5'-UTR concerns the translation of the protein encoded by the ORF. More preferably, the function comprises enhancing translation efficiency of the ORF linked to the 3'-UTR and/or the 5'-UTR or fragment or variant thereof.
  • the variant, the fragment, and the variant fragment in the context of the present invention fulfil the function of stabilizing an mRNA, preferably in a mammalian cell, such as a human cell, compared to an mRNA comprising a reference 3'- UTR and/or a reference 5'-UTR or lacking a 3'-UTR and/or a 5'-UTR, and/or the function of enhancing, stabilizing and/or prolonging protein production from an mRNA, preferably in a mammalian cell, such as in a human cell, compared to an mRNA comprising a reference 3'- UTR and/or a reference 5'-UTR or lacking a 3'-UTR and/or a 5'-UTR, and/or the function of increasing protein production from an mRNA, preferably in a mammalian cell, such as in a human cell, compared to an mRNA comprising a reference 3'-UTR and/or a reference 5'- UTR or lacking
  • a reference 3'-UTR and/or a reference 5'-UTR may be, for example, a 3'-UTR and/or a 5'-UTR naturally occurring in combination with the ORF.
  • a functional variant, a functional fragment, or a functional variant fragment of a 3'-UTR and/or a 5'-UTR of a transcript of a gene preferably does not have a substantially diminishing effect on the efficiency of translation of the mRNA which comprises such variant, fragment, or variant fragment of a 3'-UTR and/or a 5'-UTR compared to the wild-type 3'-UTR and/or the wild-type 5'-UTR from which the variant, the fragment, or the variant fragment is derived.
  • a particularly preferred function of a "functional fragment", a “functional variant” or a “functional fragment of a variant” of the 3'-UTR and/or the 5'-UTR of a transcript of a gene in the context of the present invention is the enhancement, stabilization and/or prolongation of protein production by expression of an mRNA carrying the functional fragment, functional variant or functional fragment of a variant as described above.
  • the functional fragment of the 3'-UTR and/or of the 5'-UTR preferably exhibits a length of at least about 3 nucleotides, preferably of at least about 5 nucleotides, more preferably of at least about 10, ⁇ 5, 20, 25 or 30 nucleotides, even more preferably of at least about 50 nucleotides, most preferably of at least about 70 nucleotides.
  • the 3'-UTR and/or the 5'-UTR of a transcript of a gene or a fragment or variant thereof exhibits a length of between 3 and about 500 nucleotides, preferably of between 5 and about 150 nucleotides, more preferably of between 10 and 100 nucleotides, even more preferably of between 15 and 90, most preferably of between 20 and 70.
  • the 5'- UTR moiety and/or the 3'-UTR moiety is characterized by less than 500, 400, 300, 200, 150 or less than 100 nucleotides.
  • the present invention comprises the association of such 5'-UTRs and 3'-UTRs with a nucleic acid molecule of interest, e.g. an ORF.
  • association the nucleic acid molecule or the vector with a 3'-UTR moiety and/or a 5'-UTR moiety or "associating the optimized nucleic acid molecule or the vector with a 3'-UTR moiety and/or a 5'-UTR moiety", or the like, in the context of the present invention preferably means functionally associating or functionally combining the artificial (optimized) nucleic acid molecule or the vector with the 3'-UTR moiety and/or with the 5'-UTR moiety. Thereby, further optimization (i.e. gain of additional desired functional properties) may be achieved.
  • the artificial (optimized) nucleic acid molecule and the 3'-UTR moiety and/or the 5'-UTR moiety are associated or coupled such that the function of the 3'-UTR moiety and/or of the 5'-UTR moiety, e.g., the RNA and/or protein production prolonging and/or increasing function, is exerted.
  • the 3'-UTR moiety and/or the 5'-UTR moiety is integrated into the artificial (optimized) nucleic acid molecule, preferably the mRNA molecule, 3' and/or 5', respectively, to an open reading frame (ORF), preferably immediately 3' to an open reading frame and/or immediately 5' to an open reading frame, the 3'-UTR moiety preferably between the open reading frame and a poly(A) sequence or a polyadenylation signal.
  • ORF open reading frame
  • the 3'-UTR moiety and/or the 5'- UTR moiety is integrated into the artificial (optimized) nucleic acid molecule or the vector, preferably the mRNA, as 3'-UTR and/or as 5'-UTR respectively, i.e.
  • the 3'-UTR moiety and/or the 5'-UTR moiety is the 3'-UTR and/or the 5'-UTR, respectively, of the artificial (optimized) nucleic acid molecule or the vector, preferably the mRNA, i.e., such that the 5'-UTR ends immediately before the 5'-end of the ORF and the 3'-UTR extends from the 3'-side of the open reading frame to the 5'-side of a poly(A) sequence or a polyadenylation signal, optionally connected via a short linker, such as a sequence comprising or consisting of one or more restriction sites.
  • a short linker such as a sequence comprising or consisting of one or more restriction sites.
  • the terms "associating the artificial nucleic acid molecule or the vector with a 3'-UTR moiety and/or a 5'-UTR moiety” or associating the optimized nucleic acid molecule or the vector with a 3'-UTR moiety and/or a 5'-UTR moiety” mean functionally associating the 3'-UTR moiety and/or the 5'-UTR moiety with an open reading frame located within the artificial (optimized) nucleic acid molecule or the vector, preferably within the mRNA molecule. Thereby, further optimization may be achieved.
  • the association with a 3'-UTR moiety and/or a 5'-UTR moiety can either be achieved by de novo association of individual moieties, or by modifying a pre-existing nucleic acid (template).
  • the present invention comprises a method of associating an open reading frame (ORF) encoding a polypeptide or protein of interest and optional further element(s) with a 3'-UTR moiety and/or with a 5'-UTR moiety.
  • the optimized nucleic acid of the invention comprises both (i) at least one preferred 5'-UTR and (ii) at least one preferred 3'-UTR, each as described herein.
  • the optimized nucleic acid molecule according to the present invention may comprise more than one 3'-UTR moieties and/or more than one 5'-UTR moieties as described herein.
  • the optimized nucleic acid molecule according to the present invention may comprise one, two, three, four or more 3'-UTR moieties, and/or one, two, three, four or more 5'-UTR moieties, wherein the individual 3'-UTR moieties may be the same or they may be different, and similarly, the individual 5'-UTR moieties may be the same or they may be different.
  • the optimized nucleic acid molecule according to the present invention may comprise two essentially identical 3'-UTR moieties.
  • the optimized nucleic acid molecule according to the present invention may comprise two essentially identical 5'-UTR moieties.
  • 3'-UTR moiety refers to a nucleic acid sequence which comprises or consists of a nucleic acid sequence that is derived from a 3'-UTR or from a variant or a fragment of a 3'- UTR.
  • a "3'-UTR moiety” preferably refers to a nucleic acid sequence which is comprised by a 3'-UTR of an optimized nucleic acid sequence, such as an optimized mRNA. Accordingly, in the sense of the present invention, preferably, a 3'-UTR moiety may be comprised by the 3'-UTR of an mRNA, preferably of an optimized mRNA, or a 3'-UTR moiety may be comprised by the 3'-UTR of the respective transcription template.
  • a 3'-UTR moiety is a nucleic acid sequence which corresponds to the 3'-UTR of an mRNA, preferably to the 3'-UTR of an optimized mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct.
  • a 3'-UTR moiety in the sense of the present invention functions as a 3'-UTR or codes for a nucleotide sequence that fulfils the function of a 3'-UTR.
  • 5'-UTR moiety refers to a nucleic acid sequence which comprises or consists of a nucleic acid sequence that is derived from a 5'-UTR or from a variant or a fragment of a 5'-UTR.
  • a “5'-UTR moiety” preferably refers to a nucleic acid sequence which is comprised by a 5'-UTR of an optimized nucleic acid sequence, such as an optimized mRNA.
  • a 5'-UTR moiety may be comprised by the 5'-UTR of an mRNA, preferably of an optimized mRNA, or a 5'-UTR moiety may be comprised by the 5'-UTR of the respective transcription template.
  • a 5'-UTR moiety is a nucleic acid sequence which corresponds to the 5'-UTR of an mRNA, preferably to the 5'-UTR of an optimized mRNA, such as an mRNA obtained by transcription of a genetically engineered vector construct.
  • a 5'-UTR moiety in the sense of the present invention functions as a 5'-UTR or codes for a nucleotide sequence that fulfils the function of a 5'-UTR.
  • the 3'-UTR moiety and/or the 5'-UTR moiety in the optimized nucleic acid molecule according to the present invention provides one or more beneficial UTR property to said optimized nucleic acid molecule.
  • the optimized nucleic acid molecule according to the present invention may in particular, comprise:
  • a 3'-UTR moiety which provides one or more beneficial UTR property to said optimized nucleic acid molecule and a 5'-UTR moiety which provides one or more beneficial UTR property to said optimized nucleic acid molecule.
  • said at least one 3'-UTR moiety which provides one or more beneficial UTR property to said optimized nucleic acid molecule or said at least one 5'-UTR moiety which provides one or more beneficial UTR property to said optimized nucleic acid molecule can be selected from natural ly occurring (preferably heterologous) 3'-UTR moieties and 5'-UTR moieties (together naturally occurring UTR moieties or wild-type UTR moieties), and from optimized 3'-UTR moieties and optimized 5'-UTR moieties (together optimized UTR moieties).
  • Wild-type UTR moieties can be selected from the group comprising wild-type UTR moieties published in the literature and in publically accessible databases, such as GenBank (NCBI), and wild-type UTR moieties not previously published. The latter can be identified by sequencing mRNAs found in cells, preferably mammalian cells. Using this approach, the present inventors identified several wild-type UTR moieties not previously published, and UTR moieties of this type are provided in the present invention.
  • the term artificial UTR moiety is not particularly limited and can refer to any nucleic acid sequence not found in nature, i.e. nonidentical to a wild-type UTR moiety.
  • the artificial UTR moiety used in the present invention is a nucleic acid sequence which shows a certain degree of sequence identity to a wild-type UTR moiety, such as 1 0 to 99.9 %, 20 to 99 %, 30 to 98 %, 40 to 97 %, 50 to 96 %, 60 to 95 %, 70 to 90 %.
  • artificial UTR moiety used in the present invention is identical to a wild-type UTR moiety, except that one, or two, or three, or four, or five, or more than five nucleotides have been substituted by the same number of nucleotides (e.g. one nucleotide being substituted by one nucleotide).
  • substitution of one nucleotide is a substitution by the respective complementary nucleotide.
  • Preferred artificial UTR moieties correspond to wild-type UTR moieties, except that (i) some or all ATG triplets in a wild-type 5'-UTR moiety (if present) are converted to the triplet TAG; and/or (ii) selected cleavage site(s) for a particular restriction enzyme in a wild-type 5'-UTR moiety or in a in a wild-type 3'-UTR moiety (if present) are eliminated by substituting one nucleotide within the cleavage site for said specific restriction enzyme by the complementary nucleotide, thereby removing the cleavage sites for said specific restriction enzyme.
  • UTR moiety comprises a cleavage site for said specific restriction enzyme, and when said particular restriction enzyme is (planned to be) used in subsequent cloning steps. Since such internal cleavage of 5'-UTR moieties and 3'-UTR moieties is undesired, an artificial UTR moiety can be generated in which the restriction cleavage site for said specific restriction enzyme is eliminated. Such substitution can be done by any suitable method known to the person skilled in the art, e.g. use of modified primers by PCR.
  • the optimized nucleic acid molecule according to the present invention comprises a 3'-UTR moiety which provides one or more beneficial UTR property to said optimized nucleic acid molecule and/or a 5'-UTR moiety which provides one or more beneficial UTR property to said optimized nucleic acid molecule.
  • the optimized nucleic acid molecule according to the present invention comprises at least one 3'-UTR moiety and at least one 5'-UTR moiety, i.e. at least one 3'-UTR moiety which provides one or more beneficial UTR property to said optimized nucleic acid molecule and at least one 5'-UTR moiety which provides one or more beneficial UTR property to said optimized nucleic acid molecule.
  • Specific useful UTRs useful for the present invention may be selected from the specific 5'- UTRs and the specific 3'-UTRs described in the following.
  • the 5'-UTR moiety used in the present invention differs from a wild- type 5'-UTR moiety.
  • Such 5'-UTR moieties are designated "artificial 5'-UTR moieties".
  • the artificial 5'-UTR moiety differs from the wi ld-type 5'-UTR moiety it is based on in that at least one nucleotide, such as two nucleotides, three nucleotides, four nucleotides, five nucleotides, six nucleotides, seven nucleotides, eight nucleotides, nine nucleotides, ten nucleotides, or more than ten nucleotides, is/are exchanged.
  • nucleotide exchange may be recommendable in case the wild-type 5'-UTR moiety comprises a nucleotide moiety which is considered disadvantageous.
  • a nucleotide moiety which is considered disadvantageous is selected from (i) an internal ATG triplet (i.e. an ATG triplet other than the start codon of the open reading frame of the nucleic acid of the invention) or (ii) a restriction enzyme recognition site (cleavage site), particularly the restriction enzyme recognition site (cleavage site) which is recognized (cleavable) by a restriction enzyme used in the process of making (cloning) the optimized nucleic acid of the present invention.
  • the 5'-UTR comprises or consists of a nucleic acid sequence which is derived from the 5'-UTR of a TOP gene or which is derived from a fragment, homolog or variant of the 5'-UTR of a TOP gene.
  • the nucleic acid sequence which is derived from the 5'-UTR of a TOP gene is derived from a eukaryotic TOP gene, preferably a plant or animal TOP gene, more preferably a chordate TOP gene, even more preferably a vertebrate TOP gene, most preferably a mammalian TOP gene, such as a human TOP gene.
  • the 5'-UTR is preferably selected from 5'-UTR moieties comprising or consisting of a nucleic acid sequence which is derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO.
  • the 5'-UTR comprises or consists of a nucleic acid sequence which is derived from a nucleic acid sequence extending from nucleotide position 5 (i.e. the nucleotide that is located at position 5 in the sequence) to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g. the nucleotide position immediately 5' to the ATG sequence, of a nucleic acid sequence selected from SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO. 1422 of the patent application WO2013/143700, from the homologs of SEQ ID NOs. 1 -1363, SEQ ID NO.
  • the 5'-UTR is derived from a nucleic acid sequence extending from the nucleotide position immediately 3' to the 5'TOP to the nucleotide position immediately 5' to the start codon (located at the 3' end of the sequences), e.g. the nucleotide position immediately 5' to the ATG sequence, of a nucleic acid sequence selected from SEQ ID NOs. 1 -1363, SEQ ID NO. 1395, SEQ ID NO. 1421 and SEQ ID NO.
  • the further 5'-UTR comprises or consists of a nucleic acid sequence which is derived from a 5'-UTR of a TOP gene encoding a ribosomal protein or from a variant of a 5'-UTR of a TOP gene encoding a ribosomal protein.
  • the 5'-UTR moiety comprises or consists of a nucleic acid sequence which is derived from a 5'- UTR of a nucleic acid sequence according to any of SEQ ID NOs: 170, 232, 244, 259, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1346, 1347, 1348, 1349, 1350,
  • the 5'-UTR comprises or consists of a nucleic acid sequence which is derived from a 5'-UTR of a TOP gene encoding a ribosomal Large protein (RPL) or from a homolog or variant of a 5'-UTR of a TOP gene encoding a ribosomal Large protein (RPL).
  • RPL ribosomal Large protein
  • the 5'-UTR moiety comprises or consists of a nucleic acid sequence which is derived from a 5'-UTR of a nucleic acid sequence according to any of SEQ ID NOs: SEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421 and 1422 of the patent application WO2013/143700, a corresponding RNA sequence, a homolog thereof, or a variant thereof as described herein, optionally lacking the 5'TOP motif.
  • the 5'-UTR moiety comprises or consists of a nucleic acid sequence which is derived from the 5'-UTR of a ribosomal protein Large 32 gene, preferably from a vertebrate ribosomal protein Large 32 (L32) gene, more preferably from a mammalian ribosomal protein Large 32 (L32) gene, most preferably from a human ribosomal protein Large 32 (L32) gene, or from a variant of the 5'-UTR of a ribosomal protein Large 32 gene, preferably from a vertebrate ribosomal protein Large 32 (L32) gene, more preferably from a mammalian ribosomal protein Large 32 (L32) gene, most preferably from a human ribosomal protein Large 32 (L32) gene, wherein preferably the further 5'-UTR does not comprise the 5'TOP of said gene.
  • the 5'-UTR moiety can comprise or consist of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NO: 1 804 (5'-UTR of human ribosomal protein Large 32 lacking the 5' terminal oligopyrimidine tract; corresponding to SEQ ID NO.
  • the 5'-UTR moiety comprises or consists of a fragment of a nucleic acid sequence which has an identity of at least about 40%, preferably of at least about 50%, preferably of at least about 60%, preferably of at least about 70%, more preferably of at least about 80%, more preferably of at least about 90%, even more preferably of at least about 95%, even more preferably of at least about 99% to the nucleic acid sequence according to SEQ ID NO: 1 804 or more preferably to a corresponding RNA sequence.
  • the fragment exhibits a length of at least about 20 nucleotides or more, preferably of at least about 30 nucleotides or more, more preferably of at least about 40 nucleotides or more.
  • the fragment is a functional fragment as described herein.
  • the optimized nucleic acid molecule comprises a 5'-UTR which comprises or consists of a nucleic acid sequence which is derived from the 5'-UTR of a vertebrate TOP gene, such as a mammalian, e.g.
  • a human TOP gene selected from RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS1 1 , RPS12, RPS13, RPS14, RPS15, RPS1 5A, RPS1 6, RPS1 7, RPS18, RPS1 9, RPS20, RPS21 , RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL1 1 , RPL12, RPL1 3, RPL13A, RPL14, RPL15, RPL1 7, RPL18, RPL1 8A, RPL19, RPL21 , RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, R
  • any polynucleotide moiety may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the above-described 5'-UTR sequences.
  • the 3'-UTR can comprise or consist of a nucleic acid sequence which is derived from a 3'- UTR of a gene selected from the group consisting of an albumin gene, an a-globin gene, a ⁇ - globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (I) gene, or from a variant of a 3'-UTR of a gene selected from the group consisting of an albumin gene, an a-globin gene, a ⁇ -globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1 (1) gene according to SEQ ID NO: 1369-1 390 of the patent application WO2013/143700, whose disclosure is incorporated herein by reference.
  • a collagen alpha gene such as a collagen alpha 1 (1) gene according to SEQ ID NO: 1369
  • the nucleic acid molecule of the present invention can comprises a 3'-UTR moiety derived from the nucleic acids according to SEQ ID NO. 1369-1390 of the patent application WO2013/143700 or a fragment, homolog or variant thereof.
  • the further 3'-UTR comprises or consists of a nucleic acid sequence which is derived from a 3'-UTR of an albumin gene, preferably a vertebrate albumin gene, more preferably a mammalian albumin gene, most preferably a human albumin gene according to SEQ ID NO: 1 728 (Human albumin 3'-UTR; corresponding to SEQ ID NO: 1369 of the patent application WO2013/143700).
  • the 3'-UTR may comprise a nucleic acid sequence derived from a fragment of the human albumin gene according to SEQ ID NO: 1 729 (albumin7 3'-UTR; corresponding to SEQ ID NO: 1 376 of the patent application WO2013/143700).
  • the 3'-UTR of the optimized nucleic acid molecule comprises consists of the nucleic acid sequence according to SEQ ID NO: 1 729, or a correspond RNA sequence.
  • the 3'-UTR may also comprise or consist of a nucleic acid sequence derived from a ribosomal protein coding gene, whereby ribosomal protein coding genes from which a further 3'-UTR may be derived include, but are not limited to, ribosomal protein L9 (RPL9), ribosoma protein L3 (RPL3), ribosomal protein L4 (RPL4), ribosomal protein L5 (RPL5), ribosoma protein L6 (RPL6), ribosomal protein L7 (RPL7), ribosomal protein L7a (RPL7A), ribosoma protein L1 1 (RPL1 1 ), ribosomal protein L12 (RPL12), ribosomal protein L13
  • RPL9 ribosomal protein L9
  • RPL3 ribosoma protein L3
  • RPL4 ribosomal protein L4
  • RPL5 ribosomal protein L5
  • RPL6
  • RPL13 ribosomal protein L23
  • RPL18 ribosomal protein L18
  • RPL18A ribosoma protein L1 8a
  • RPL1 9 ribosomal protein L21
  • RPL21 ribosomal protein L22
  • RPL23A ribosomal protein L23a
  • RPL1 7 ribosomal protein L24
  • RPL26 ribosomal protein L26
  • RPL26 ribosomal protein L27
  • RPL30 ribosomal protein L30
  • RPL27A ribosomal protein L28
  • RPL28 ribosomal protein L29
  • RPL29 ribosomal protein L31
  • RPL32 ribosomal protein L32
  • RPL35A ribosomal protein L37
  • RPL37a ribosomal protein L37a
  • RPL37A ribosomal protein L38 (RPL38), ribosomal protein L39 (RPL39), ribosoma protein, large, P0 (RPLPO), ribosomal protein, large, PI (RPLP1 ), ribosoma protein, large, P2 (RPLP2), ribosomal protein S3 (RPS3), ribosomal protein S3A
  • RPS3A ribosomal protein S4, X- linked (RPS4X), ribosomal protein S4, Y-linked 1 (RPS4Y1 ), ribosoma protein S5 (RPS5), ribosomal protein S6 (RPS6), ribosomal protein S7 (RPS7), ribosoma protein S8 (RPS8), ribosomal protein S9 (RPS9), ribosomal protein S10 (RPS10), ribosoma protein S1 1 (RPS1 1 ), ribosomal protein S12 (RPS12), ribosomal protein S13
  • RPS20 ribosomal protein S21
  • RPS23 ribosomal protein S23
  • RPS25 ribosoma protein S25
  • RS26 ribosomal protein S26
  • RPS27 ribosomal protein S27a
  • RPS28 ribosomal protein S28
  • RPS29 ribosomal protein L1 5
  • RPL2 ribosomal protein S2
  • RPL14 ribosomal protein S14
  • RPL14 ribosomal protein L10
  • RPL1 0 ribosomal protein L10a
  • RPL35 ribosomal protein L1 3a
  • RPL1 3A ribosomal protein L36
  • RPL36 ribosomal protein L36a
  • RPL36A ribosomal protein L41
  • RPL41 ribosomal protein S1 8
  • RPL8 ribosomal protein L8
  • RPL34 RPL
  • the 3'-UTR comprises or consists of a nucleic acid sequence according to any one of SEQ ID NOs: 1 0 to 205 of WO201 5/1 01 414.
  • the at least one 3'-untranslated region element (3'-UTR element) comprises or consists of a nucleic acid sequence which is derived from the 3'-UTR of a FIG4 gene or from a variant of the 3'-UTR of a FIG4 gene.
  • the term "a FIG4 gene” generally refers to a gene encoding FIG4, which is also known as, for instance, Sac Domain-Containing Inositol Phosphatase 3, SAC3, S.
  • FIG4 gene refers to any FIG4 gene, irrespective of the species, from which it is derived.
  • FIG4 gene refers to a mammalian F1G4 gene.
  • a FIG4 gene comprises any paralogs and orthologs of a mammalian FIG4 gene.
  • any sequence, which is characterized by substantial sequence simi larity or identity is referred to as FIG4 gene in the context of the present invention.
  • Fig4 genes and corresponding 3'-UTRs are disclosed in WO201 5/1 01 41 5, the contents of which are herein incorporated by reference.
  • any 3'-UTR of a FIG4 gene according to the disclosure of WO201 5/1 0141 5 can be selected as a nucleic acid moiety according to the present invention.
  • Preferred moieties are represented by SEQ ID NO: 1 and 2 of WO2015/10141 5. This reference is incorporated herein in its entirety.
  • any polynucleotide moiety may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the above-described 3'-UTR sequences.
  • a miRNA may also be selected as a moiety in the present invention. Any miRNA moiety known in the art may be selected. Such a moiety can be selected from microRNA target sequences, microRNA seqences, or microRNA seeds. For example, miRNA sequences (microRNA target sequences, microRNA seqences, or microRNA seeds) are described in WO 2015085318 A2, US 2005/026121 8 and US2005/00590O5.
  • microRNAs are 19-25 nucleotide long noncoding RNAs. miRNAs bind to 3'-UTR of nucleic acid molecules. This causes down-regulation of gene expression, either by reducing nucleic acid molecule stability or by inhibiting translation.
  • the polynucleotides of the present invention may comprise one or more microRNA target sequences, microRNA seqences, or microRNA seeds.
  • microRNA site refers to a polynucleotide sequence to which a microRNA can bind or otherwise associate, "binding" typically occurs by Watson-Crick hybridization; but any otherwise stable association of the microRNA with the target sequence at or adjacent to the microRNA site is also comprised in the concept of a "microRNA site” according to the present invention.
  • a microRNA sequence comprises a "seed" region, i.e., a sequence typically in the region of positions 2-8 of a mature microRNA.
  • the seed region sequence has perfect Watson- Crick complementarity to the miRNA target sequence.
  • Such a microRNA seed may comprise positions 2-8, or alternatively 2-7 of the mature microRNA.
  • a microRNA seed comprises 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1 .
  • A adenine
  • a microRNA seed comprises 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed- complementary site in the corresponding miRNA target is flanked byan adenine (A) opposed to microRNA position 1 .
  • A an adenine
  • Respective nucleic acid modules are disclosed in Grimson et al.; Mol Cell. 2007 Jul 6;27(1 ):91 -1 05
  • a microRNA target sequence is typically designed to be comprised in a 3 '-UTR or otherwise 3' (upstream) of an open reading frame.
  • the miRNA target sequence is thought to target the molecule for degradation or reduced translation, provided that a corresponding microRNA in question is available. This allows to control any undesired off-target effects upon delivery of the nucleic acid molecule of the present invention.
  • miR-122 a microRNA abundant in liver, can inhibit the expression of the nucleic acid of the present invention, if one or multiple target sites of miR-122 are present (e.g.
  • microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they occur, e.g., in order to increase protein expression in specific tissues.
  • one or more mi R-122 binding sites may be removed to improve protein expression in the liver.
  • microRNAs are known to regulate mRNA, and thereby protein expression, without limitation in liver (miR- 122), heart (miR-lcl, miR-149), endothelial cells (miR- 1 7-92, miR-126), adipose tissue (let-7, miR-30c), kidney (miR-192, miR-1 94, miR-204), myeloid cells (miR-142-3p, miR-142-5p, miR-1 6, miR-21 , miR-223, miR-24, miR-27),muscle (miR-133, miR-206, miR-208), and lung epithelial cells (let-7, miR-133, miR-126).
  • MicroRNA can also regulate complex biological processes such as angiogenesis (miR-132) (e.g. Anand and Cheresh, Curr. Opin. Hematol. 201 1 , 18: 1 71 -1 76;).
  • binding sites for microRNAs may be removed or introduced, in order to tailor the expression of the polynucleotides expression to desired cell types or tissues, or to the context of relevant biological processes.
  • Listings of miRNA sequences and binding sites areavailable to the public. Any sequence disclosed in the literature discussed herein may be used in the context of the present invention: examples of microRNA that drive tissue- or disease-specific gene expression are listed in Getner and Naldini, Tissue Antigens.
  • microRNA seed sites are incorporation of miR-142 sites into a UGT1 A1 - expressing lentiviral vector, which causes reduced expression in antigen-presentating cells, leading to the absence of an immune response against the virally expressed UGT1 A1 as disclosed in Schmitt et al., Gastroenterology 2010; 139:999-1007; Gonzalez-Asequinolaza et al. Gastroenterology 2010, 139:726-729.
  • incorporation of one or more miR-142 seed sites into mRNA is thought to be important in the case of treatment of patients with complete protein deficiencies (UGT1 A1 type I, LDLR-deficient patients, CRIM-negative Pompe patients, etc.).
  • the nucleic acid molecule of the present invention can be designed to fit such purposes.
  • Any polynucleotide may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of such miRNA sequences.
  • the present invention allows to specifically design polynucleotide molecules for targeted expression in specific cell types, or under specific biological conditions.
  • polynucleotides can be designed that for protein expression in a tissue or in the context of a biological condition.
  • IRES An internal ribosome entry site, abbreviated IRES, is a nucleotide sequence that allows for translation initiation in the middle of a messenger RNA (mRNA) sequence as part of the greater process of protein synthesis. While translation in eukaryotes is usually initiated only at the 5' end of the mRNA molecule, presence of an IRES allows translation of the RNAs in a cap-independent manner. In nature, it is common that IRESes are located in the 5'UTR of RNA viruses.
  • mRNA messenger RNA
  • Presence of an IRES can allow for translation of two proteins from a single transcript (RNA): for such purposes, part of the present invention, an IRES is present downstream of the coding region of a first polypeptide element, but upstream of the coding region of a second polypeptide element on the same transcript. Translation of the first coding region is initiated at the normal 5' cap, and the translation of the second coding region at the IRES.
  • IRES allow the expression of multiple proteins from a single nucleic acid molecule.
  • an internal ribosome entry site (IRES) sequence or IRES-motif may separate several open reading frames, for example if the optimized nucleic acid molecule encodes for two or more peptides or proteins.
  • An IRES-sequence may be particularly helpful if the optimized nucleic acid molecule is a bi- or multicistronic nucleic acid molecule.
  • such IRES are particularly useful when present in a nucleic acidencoding at least two functional protein moieties, such as at least one polypeptide or protein of interest and at least one further polypeptide or protein element, preferably also selected from the list of coding moieties of the present invention.
  • the IRES is typically located on the polynucleotide chain in between the coding region for the protein of interest and the coding region for the at least one further protein element, so that translation leads to two separate polypeptide molecules, at least one of them being a polypeptide or protein of interest.
  • nucleotide sequence of the IRES used in the present invention is selected from the following list of nucleotide sequences (SEQ ID NOs: 1 566-1 662).
  • any polynucleotide may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the IRES sequences SEQ ID NOs: 1 566-1 662.
  • the optimized nucleic acid molecule may additionally comprise a histone stem- loop.
  • a histone-stem-loop if present, is preferably localized 3' (downstream) of a 3'UTR moiety, and upstream of a poly(A) sequence or polyadenylation signal (if present).
  • an optimized nucleic acid molecule according to the present invention may, for example, comprise in 5'-to-3'-direction an ORF encoding a polypeptide of interest and optionally further element(s), a 3'-UTR moiety, an optional histone stem-loop sequence, an optional poly(A) sequence or polyadenylation signal and an optional poly(C) sequence.
  • the optimized nucleic acid molecule according to the present invention may, for example, comprise in 5'-to-3'-direction an 5'-UTR moiety, an ORF encoding a polypeptide of interest and optional ly further element(s), an optional histone stem-loop sequence, an optional poly(A) sequence or polyadenylation signal and an optional poly(C) sequence.
  • the optimized nucleic acid molecule according to the present invention may, for example, comprise in 5'-to-3'-direction an 5'-UTR moiety, an ORF encoding a polypeptide of interest and optionally further element(s), a 3'-UTR moiety, an optional histone stem-loop sequence, an optional poly(A) sequence or polyadenylation signal and an optional poly(C) sequence.
  • It may also comprise in 5'-to-3'-direction an ORF, an 3'-UTR moiety, an optional poly(A) sequence, an optional poly (C) sequence and an optional histone stem-loop sequence, or in 5'-to-3'-direction an 5'-UTR moiety, an ORF, an optional poly(A) sequence, an optional poly(C) sequence and an optional histone stem-loop sequence, or in 5'-to-3'- direction an 5'-UTR element, an ORF, a 3'-UTR element, an optional poly(A) sequence, an optional poly(C) sequence and an optional histone stem-loop sequence.
  • the optimized nucleic acid molecule according to the invention further comprises at least one histone stem-loop sequence.
  • histone stem-loop sequences are preferably selected from histone stem-loop sequences as disclosed in WO 2012/01 9780, whose disclosure is incorporated herewith by reference.
  • histone stem-loop sequences are preferably selected from histone stem- loop sequences as disclosed in WO 201 3/143699, whose disclosure is incorporated herewith by reference
  • a histone stem-loop sequence suitable to be used within the present invention, is preferably selected from at least one of the following formulae (I) or (II):
  • N1-6 [N0-2GN3-5] [No- 4 (U/T)No-4] [N3-5CN0-2] N1-6
  • steml or stem2 bordering elements Ni-6 is a consecutive sequence of 1 to 6, preferably of 2 to 6, more preferably of 2 to 5, even more preferably of 3 to 5, most preferably of 4 to 5 or 5 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C, or a nucleotide analogue thereof;
  • steml [N0-2GN 3 -5] is reverse complementary or partially reverse complementary with element stem2, and is a consecutive sequence between of 5 to 7 nucleotides; wherein N0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1 , more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof; wherein N3.5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N,
  • U/T represents uridine, or optionally thymidine
  • stem2 [N3-5CN0 is reverse complementary or partially reverse complementary with element steml , and is a consecutive sequence between of 5 to 7 nucleotides
  • N 3 . 5 is a consecutive sequence of 3 to 5, preferably of 4 to 5, more preferably of 4 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G and C or a nucleotide analogue thereof
  • N0-2 is a consecutive sequence of 0 to 2, preferably of 0 to 1 , more preferably of 1 N, wherein each N is independently from another selected from a nucleotide selected from A, U, T, G or C or a nucleotide analogue thereof
  • C is cyticline or an analogue thereof, and may be optionally replaced by a guanosine or an analogue thereof provided that its complementary nucleoside guanosine in steml is replaced by
  • steml and stem2 are capable of base pairing with each other forming a reverse complementary sequence, wherein base pairing may occur between steml and stem2, e.g. by Watson-Crick base pairing of nucleotides A and UAT or G and C or by non-Watson-Crick base pairing e.g. wobble base pairing, reverse Watson-Crick base pairing, Hoogsteen base pairing, reverse Hoogsteen base pairing or are capable of base pairing with each other forming a partially reverse complementary sequence, wherein an incomplete base pairing may occur between stemi and stem2, on the basis that one or more bases in one stem do not have a complementary base in the reverse complementary sequence of the other stem.
  • the histone stem-loop sequence may be selected according to at least one of the following specific formulae (la) or (Ma): formula (la) (stem-loop sequence without stem bordering elements):
  • N, C, G, T and U are as defined above.
  • the optimized nucleic acid molecule sequence may comprise at least one histone stem-loop sequence according to at least one of the following specific formulae (lb) or (lib): formula (lb) (stem-loop sequence without stem bordering elements):
  • stemi loop stem2 formula (lib) stem-loop sequence with stem bordering elements
  • N, C, G, T and U are as defined above.
  • a particular preferred histone stem-loop sequence is the sequence according to SEQ ID NO: 1 731 , or more preferably the corresponding RNA sequence.
  • the optimized nucleic acid molecule according to the present invention may further comprise optionally a 5'-cap.
  • the optional 5'-cap is preferably located 5' to the ORF, more preferably 5' to the at least one 5'-UTR (if present) within the optimized nucleic acid molecule according to the present invention. This embodiment is particularly useful when the nucleic acid molecule is an RNA molecule.
  • a 5'-cap may be added in a cell, or may alternatively be added in vitro. Further details of a 5'-cap useful in the present invention are described below in the context of chemical modifications.
  • the optimized nucleic acid molecule according to the present invention further comprises a poly(A) sequence and/or a polyadenylation signal.
  • a poly(A) sequence is particularly useful when the nucleic acid molecule is an RNA molecule, and is preferably present in an RNA molecule comprising a 3'-UTR.
  • the poly(A) sequence is located 3' to the 3'-UTR moiety, more preferably the poly(A) sequence is connected to the 3'-end of a 3'-UTR moiety. The connection may be direct or indirect, for example, via a stretch of 2, 4, 6, 8, 10, 20 etc.
  • nucleotides such as via a linker of 1 -50, preferably of 1 -20 nucleotides, e.g. comprising or consisting of one or more restriction sites.
  • linker of 1 -50 preferably of 1 -20 nucleotides, e.g. comprising or consisting of one or more restriction sites.
  • the optimized nucleic acid molecule according to the present invention does not comprise a 3'-UTR, for example if it only comprises at least one 5'-UTR moiety, it preferably still comprises a poly(A) sequence and/or a polyadenylation signal.
  • a DNA molecule comprising an ORF, optionally followed by a 3' UTR may contain a stretch of thymidine nucleotides which can be transcribed into a poly(A) sequence in the resulting mRNA.
  • the length of the poly(A) sequence may vary.
  • the poly(A) sequence may have a length of about 20 adenine nucleotides up to about 300 adenine nucleotides, preferably of about 40 to about 200 adenine nucleotides, more preferably from about 50 to about 100 adenine nucleotides, such as about 60, 70, 80, 90 or 100 adenine nucleotides.
  • the nucleic acid of the invention comprises a poly(A) sequence of about 60 to about 70 nucleotides, most preferably 64 adenine nucleotides.
  • the optional polyadenylation signal is located downstream of the 3' of the 3'-UTR moiety.
  • consensus sequence may be recognised by most animal and bacterial cell-systems, for example by the polyadenylation-factors, such as cleavage/polyadenylation specificity factor (CPSF) cooperating with CstF, PAP, PAB2, CFI and/or CFII.
  • CPSF cleavage/polyadenylation specificity factor
  • the polyadenylation signal preferably the consensus sequence NNUANA
  • the polyadenylation signal is located less than about 50 nucleotides, more preferably less than about 30 bases, most preferably less than about 25 bases, for example 21 bases, downstream of the 3'-end of the 3'-UTR moiety or of the ORF, if no 3'-UTR moiety is present.
  • an optimized nucleic acid molecule according to the present invention e.g. of an artificial DNA molecule, comprising a polyadenylation signal downstream of the 3'- UTR moiety (or of the ORF) will result in a premature-RNA containing the polyadenylation signal downstream of its 3'-UTR moiety (or of the ORF).
  • the inventive optimized nucleic acid molecule may be a DNA molecule comprising a 3'-UTR moiety as described above and a polyadenylation signal, which may result in polyadenylation of an RNA upon transcription of this DNA molecule.
  • a resulting RNA may comprise a combination of a 3'-UTR moiety and a poly(A) sequence,
  • transcription of an optimized nucleic acid molecule according to the invention e.g. transcription of an optimized nucleic acid molecule comprising an open reading frame, a 3'-UTR moiety and/or a 5'-UTR moiety and optionally a polyadenylation- signal, may result in an mRNA molecule comprising an open reading frame, a 3'-UTR moiety and optionally a poly(A) sequence.
  • the invention also provides an optimized nucleic acid molecule, which is an mRNA molecule comprising an open reading frame, a 3'-UTR moiety as described above and/or a 5'-UTR moiety as described above and optionally a poly(A) sequence.
  • the 3'-UTR of the optimized nucleic acid molecule according to the invention does not comprise a polyadenylation signal or a poly(A) sequence. Further preferably, the optimized nucleic acid molecule according to the invention does not comprise a polyadenylation signal or a poly(A) sequence. More preferably, the 3'-UTR of the optimized nucleic acid molecule, or the inventive optimized nucleic acid molecule as such, does not comprise a polyadenylation signal, in particular it does not comprise the polyadenylation signal AAU/TAAA. 3.2.7 Additional Modules
  • the optimized nucleic acid molecule may comprise additional 5'-moieties, preferably a promoter or a promoter containing-sequence.
  • the promoter may drive and or regulate transcription of the optimized nucleic acid molecule according to the present invention, for example of an artificial DNA-molecule according to the present invention.
  • any polynucleotide may be selected which is characterized by at least 80 % identity, at least 85 % identity, preferably at least 90 % identity, and more preferably at least 95 % identity to any of the promoter sequences.
  • the optimized nucleic acid molecule according to the present invention may further comprise at least one hairpin moiety.
  • Hairpin moieties can support RNA folding, protect mRNA from degradation, or serve as a recognition motif for RNA binding proteins etc. Hairpin moieties may be derived from naturally occurring hairpin structures (e.g., as present in UTR regions).
  • RNA-binding proteins are proteins that bind to single stranded RNA in cells and participate in forming ribonucleoprotein complexes.
  • RBPs contain various structural motifs, such as RNA recognition motif (RRM), dsRNA binding domain, zinc finger and others.
  • RBPs have crucial roles in various cellular processes such as: cellular function, transport and localization. They especially play a major role in post-transcriptional control of RNAs, such as: splicing, polyadenylation, mRNA stabilization, mRNA localization and translation.
  • Such moieties may be incorporated into the optimized nucleic acid to increase the translation rate of the construct into protein. Furthermore, moieties for RNA binding proteins may be introduced into the optimized nucleic acid to increase the cellular stability of the construct. Such optimized nucleic acids may have a prolonged half-life which may result in a prolonged protein expression in vivo.
  • the optimized nucleic acid molecule according to the present invention may further comprise a moiety that prevents 3'-5' degradation.
  • Such moieties may comprise tailored oligonucleotides, potentially comprising modified nucleotides.
  • Such moiety that prevents 3'-5' degradation may be incorporated into an optimized nucleic acid to increase the cellular stability of the construct.
  • Such optimized nucleic acids may have a prolonged half- life which may result in a prolonged protein expression in vivo.
  • the optimized nucleic acid molecule according to the present invention may further comprise moieties that regulate RNA decay rates.
  • AU-rich elements located on the 3'UTR of mRNAs modulate mRNA stability, both as stabilizing and destabilizing elements.
  • Elements that destabi lize the optimized nucleic acid construct may be introduced into the RNA for application where a fast decay of the RNA is desired, e.g., when the expression of the encoded target protein has to be restricted.
  • Elements that stabilize the optimized nucleic acid construct may be introduced into the RNA for application where a decay of the RNA is not desired, e.g., when the expression of the encoded target protein has to be prolonged.
  • the nucleic acid molecule of the present invention may be modified in that a molecular entity (building block) found in a respective starting nucleic acid (e.g. wild-type nucleic acid) is replaced by a different molecular entity (building block).
  • a building block or molecular entity may be a deoxyribonucleotide or ribonucleotide or non-naturally occurring nucleotide.
  • the replacing molecular entity may be a different - natural ly occurring - deoxyribonucleotide or ribonucleotide, e.g. A, C, G, T, U.
  • Embodiments of replacement by a different - but naturally occurring - molecular entity include G/C modification and codon optimization, each as described below.
  • the replacing molecular entity may be a synthetic entity.
  • Embodiments include the chemical modifications described below. 4.1 Substitution by naturally occurring molecular entities
  • the replacing molecular entity may be a naturally occurring deoxyribonucleotide or ribonucleotide, so that one naturally occurring nucleotide is replaced by a different deoxyribonucleotide or ribonucleotide.
  • Embodiments thereof include G/C modification and codon optimization:
  • the optimized nucleic acid molecule according to the present invention is at least partially G/C modified.
  • the G/C content of the open reading frame of an optimized nucleic acid molecule according to the present invention may be increased compared to the G/C content of the open reading frame of a corresponding wild-type sequence, preferably by taking advantage of the degeneration of the genetic code.
  • the amino acid sequence (polypeptide or protein) encoded by the optimized nucleic acid molecule is preferably not altered, despite the G/C modification.
  • the codons of the coding sequence or the whole optimized nucleic acid molecule e.g.
  • an mRNA may therefore be varied compared to the wild-type coding sequence, such that they include an increased amount of G/C nucleotides while the translated amino acid sequence is maintained. Due to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), it is feasible to alter codons while not altering the encoded peptide/protein sequence (so-called alternative codon usage). Hence, it is possible to specifically introduce certain codons (in exchange for the respective wild-type codons encoding the same amino acid), which are more favourable with respect to stability of RNA and/or with respect to codon usage in a subject (so-called codon optimization).
  • nucleic acid sequence e.g. the open reading frame
  • codons which contain exclusively G or C nucleotides
  • no modification of the codon is necessary.
  • the codons for Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require no modification, since no A or U/T is present.
  • codons which contain A and/or U T nucleotides may be modified by substitution of other codons which code for the same amino acids but contain no A and/or U/T.
  • the codons for Pro can be modified from CC(U/T) or CCA to CCC or CCG;
  • the codons for Arg can be modified from CG(U T) or CGA or AGA or AGG to CGC or CGG;
  • the codons for Ala can be modified from GC(U/T) or GCA to GCC or GCG;
  • the codons for Gly can be modified from GG(U/T) or GGA to GGC or GGG.
  • a or (U/T) nucleotides cannot be eliminated from the codons, it is however possible to decrease the A and (UAT) content by using codons which contain a lower content of A and/or (U/T) nucleotides. Examples of these are:
  • the codons for Phe can be modified from (U/T) (U T) (U/T) to (U/T) (U T)C;
  • the codons for Leu can be modified from (U/T) (U T)A, (U/T) (U T)G, C(U T) (U T) or C(UAT)A to C(U/T)C or C(U/T)G;
  • the codons for Ser can be modified from (U/T)C(U T) or (U/T)CA or AG(U/T) to (U T)CC, (U DCG or AGC;
  • the codon for Tyr can be modified from (U/T)A(U T) to (UAT)AC;
  • the codon for Cys can be modified from (U/T)G(U/T) to (U/T)GC;
  • the codon for His can be modified from CA(U/T) to CAC;
  • the codon for Gin can be modified from CAA to CAG;
  • codons for lie can be modified from A(UiT)( ⁇ / ) or A(U/T)A to A(U/T)C;
  • codons for Thr can be modified from AQU/T) or ACA to ACC or ACG;
  • the codon for Asn can be modified from AA(UAT) to AAC;
  • the codon for Lys can be modified from AAA to AAG;
  • the codons for Val can be modified from G(U/T)(U/D or G(U T)A to G(U T)C or G(U/T)G; the codon for Asp can be modified from GA(U/T) to GAC;
  • the codon for Glu can be modified from GAA to GAG;
  • the stop codon (UAT)AA can be modified to (U/T)AG or (U/T)GA.
  • the G/C content of the open reading frame of the optimized nucleic acid molecule of the invention as defined herein is increased by at least 7%, more preferably by at least 1 5%, particularly preferably by at least 20%, compared to the G/C content of the wild-type coding region without altering the encoded amino acid sequence, i.e. using the degeneracy of the genetic code.
  • the G/C content of the open reading frame of the inventive optimized nucleic acid molecule as defined herein is particularly preferable to increase the G/C content of the open reading frame of the inventive optimized nucleic acid molecule as defined herein, to the maximum (i.e. 100% of the substitutable codons), compared to the wild-type open reading frame, without altering the encoded amino acid sequence.
  • the open reading frame is preferably at least partially codon-optimized. Codon- optimization is based on the finding that the translation efficiency may be determined by a different frequency in the occurrence of transfer RNAs (tRNAs) in cells. Thus, if so-called "rare codons" are present in the coding region of the optimized nucleic acid molecule as defined herein, to an increased extent, the translation of the corresponding modified nucleic acid sequence is less efficient than in the case where codons coding for relatively "frequent" tRNAs are present.
  • tRNAs transfer RNAs
  • the open reading frame of the optimized nucleic acid molecule is preferably modified compared to the corresponding wild-type coding region such that at least one coclon of the wild-type sequence which codes for a tRNA which is relatively rare in the cell is exchanged for a codon which codes for a tRNA which is comparably frequent in the cell and carries the same amino acid as the relatively rare tRNA.
  • the open reading frame of the optimized nucleic acid molecule as defined herein is modified such that codons for which frequently occurring tRNAs are available may replace codons which correspond to rare tRNAs.
  • all codons of the wild-type open reading frame which code for a rare tRNA may be exchanged for a codon which codes for a tRNA which is more frequent in the cell and which carries the same amino acid as the rare tRNA.
  • Which tRNAs occur relatively frequently in the cell and which, in contrast, occur relatively rarely is known to a person skilled in the art; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001 , 1 1 (6): 660-666.
  • the open reading frame is codon-optimized, preferably with respect to the system in which the optimized nucleic acid molecule according to the present invention is to be expressed, preferably with respect to the system in which the optimized nucleic acid molecule according to the present invention is to be translated.
  • the codon usage of the open reading frame is codon-optimized according to mammalian codon usage, more preferably according to human codon usage.
  • the open reading frame is codon-optimized and G/C-content modified.
  • the polynucleotide of the present invention can comprise one or more chemical modification(s).
  • the chemical modification is present as an alternative or in addition (preferably in addition) to the modules of the optimized nucleic acid molecule as described above.
  • Chemical modification generally, refers to a modified nucleotide, so that a modified nucleotide is a structural feature of such optimized nucleic acid molecule.
  • at least one nucleotide of a nucleic acid molecule e.g. deoxyribonucleic acid molecule or ribonucleic acid molecule
  • chemical modification is introduced into a nucleic acid molecule by incorporating a chemically modiefied building block at the stage of synthesizing ⁇ in vivo o in vitro) the respective nucleic acid molecule.
  • the optimized nucleic acid molecule may further comprise modifications, such as backbone modifications, sugar modifications and/or base modifications, e.g., lipid- modifications or the like.
  • modifications such as backbone modifications, sugar modifications and/or base modifications, e.g., lipid- modifications or the like.
  • modification as used herein with regard to the optimized nucleic acid molecule may refer to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications.
  • the optimized nucleic acid molecule may contain nucleotide analogues/modifications, e.g. backbone modifications, sugar modifications or base modifications.
  • a backbone modification in connection with the present invention is a modification, in which phosphates of the backbone of the nucleotides contained in a nucleic acid molecule as defined herein are chemically modified.
  • a sugar modification in connection with the present invention is a chemical modification of the sugar of the nucleotides of the nucleic acid molecule as defined herein.
  • a base modification in connection with the present invention is a chemical modification of the base moiety of the nucleotides of the nucleic acid molecule of the nucleic acid molecule.
  • nucleotide analogues or modifications are preferably selected from nucleotide analogues which are applicable for transcription and/or translation.
  • the transcription and/or the translation of the optimized nucleic acid molecule according to the present invention is not significantly impaired by the modifications.
  • nucleotide analogues are defined as natively and non- natively occurring variants of the naturally occurring nucleotides adenosine, cytosine, thymidine, guanosine and uridine. Accordingly, analogues are e.g.
  • each component of the naturally occurring nucleotide may be modified, namely the base component, the sugar (ribose or deoxyribose) component and/or the phosphate component forming the backbone (see above) of the nucleic acid molecule.
  • Analogues of guanosine, uridine, adenosine, thymidine and cytosine include, without implying any limitation, any natively occurring or non-natively occurring guanosine, uridine, adenosine, thymidine or cytosine that has been altered e.g.
  • analogue as described above, particular preference may be given according to certain embodiments of the invention to those analogues that increase the protein expression of the encoded peptide or protein or that increase the immunogenicity of the optimized nucleic acid molecule of the invention and/or do not interfere with a further modification of the optimized nucleic acid molecule that has been introduced.
  • the optimized nucleic acid molecule according to the invention may further comprise one or more of the modifications described in the following: 4.2.1 .
  • modified nucleosides and nucleotides which may be incorporated into the optimized nucleic acid molecule, preferably an RNA, as described herein, can be modified in the sugar moiety.
  • the 2 ' hydroxyl group (OH) of an RNA molecule can be modified or replaced with a number of different "oxy" or "deoxy” substituents.
  • R H, alkyl, cycloalkyl
  • “Deoxy” modifications include hydrogen, amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and O.
  • the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose.
  • a modified nucleic acid molecule can include nucleotides containing, for instance, arabinose as the sugar.
  • the phosphate backbone may further be modified in the modified nucleosides and nucleotides, which may be incorporated into the optimized nucleic acid molecule, preferably an RNA, as described herein.
  • the phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent.
  • the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate as described herein.
  • modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters.
  • Phosphorodithioates have both non-linking oxygens replaced by sulfur.
  • the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene-phosphonates).
  • modified nucleosides and nucleotides which may be incorporated into the optimized nucleic acid molecule, preferably an RNA molecule, as described herein, can further be modified in the nucleobase moiety.
  • nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil.
  • the nucleosides and nucleotides described herein can be chemically modified on the major groove face.
  • the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
  • the nucleotide analogues/modifications are selected from base modifications, which are preferably selected from 2-amino-6-chloropurineriboside-5'-triphosphate, 2-Aminopurine-riboside-5'- triphosphate; 2-aminoadenosine-5 '-triphosphate, 2'-Amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5 '-triphosphate, 2-thiouridine-5 '-triphosphate, 2'-Fluorothymidine-5'- triphosphate, 2'-0-Methyl inosine-5'-triphosphate 4-thiouridine-5'-triphosphate, 5- aminoallylcytidine-5 '-triphosphate, 5-aminoallyluridine-5 '-triphosphate, 5-bromocytidine- 5 '-triphosphate, 5-bromouridine-5 '-triphosphate, 5-Bromo-2'-
  • nucleotides for base modifications selected from the group of base- modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'- triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate.
  • modified nucleosides include pyridin-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl-pseudouridine, 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5-taurinomethyluridine, 1 -taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1 -methyl-pseudouridine, 4-thio- 1 -methyl-pseudouridine, 2-thio-l-methyl- pseudouridine, 1 -methyl- 1 -deaza-pse
  • modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3- methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1 -methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4- thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2- thio-
  • modified nucleosides include 2-aminopurine, 2, 6-diaminopurine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2- aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 - methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-di
  • modified nucleosides include inosine, 1 -methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7- deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl- guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine.
  • the nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uraci l with a methyl group or a halo group.
  • a modified nucleoside is 5 '-0-(1 -Thiophosphate)-Adenosine, 5 '-0- (l -Thiophosphate)-Cytidine, 5'-0-(1 -Thiophosphate)-Guanosine, 5 '-0-(1 -Thiophosphate)- Uridine or 5 '-0-(1 -Thiophosphate)-Pseudouridine.
  • the optimized nucleic acid molecule may comprise nucleoside modifications selected from 6-aza-cytidine, 2-th io- cytidine, alpha-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1 - methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio-uridine, 4-thio-uridine, 6-aza-uridi ne, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha-thio- guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine, 7-deaza-guanosine, N1 -methyl-adenosine, 2-amino-6-Chloro-purine
  • the optimized nucleic acid molecule preferably an RNA, as defined herein can contain a lipid modification.
  • a lipid-modified RNA typically comprises an RNA as defined herein.
  • Such a lipid-modified RNA molecule as defined herein typically further comprises at least one l inker covalently linked with that RNA molecule, and at least one lipid covalently linked with the respective linker.
  • the lipid-modified RNA molecule comprises at least one RNA molecule as defined herein and at least one (bifunctional) lipid covalently linked (without a linker) with that RNA molecule.
  • the lipid-modified RNA molecule comprises an optimized nucleic acid molecule, preferably an RNA molecule, as defined herein, at least one linker covalently li nked with that RNA molecule, and at least one lipid covalently linked with the respective linker, and also at least one (bifunctional) lipid covalently linked (without a linker) with that RNA molecule.
  • the lipid modification is present at the terminal ends of a linear RNA sequence.
  • the optimized nucleic acid molecule preferably an RNA molecule, as defined herein, can be modified by the addition of a so-called "5' cap” structure.
  • a 5'-cap is an entity, typically a modified nucleotide entity, which generally “caps" the 5'- end of a mature mRNA.
  • a 5 '-cap may typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide.
  • the 5'-cap is linked to the 5'- terminus via a 5 '-5 '-triphosphate linkage.
  • a 5'-cap may be methylated, e.g.
  • m7GpppN wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap, typically the 5'-end of an RNA.
  • m7GpppN is the 5'-cap structure which naturally occurs in mRNA transcribed by polymerase II and is therefore not considered as modification comprised in the modified RNA according to the invention.
  • the optimized nucleic acid molecule, preferably an RNA molecule, according to the present invention may comprise an m7GpppN as 5'-cap, but additionally the optimized nucleic acid molecule, preferably an RNA molecule, comprises at least one further modification as defined herein.
  • 5'cap structures include glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, 1 -(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1 ,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3'-3'- inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3 '-2 '-inverted nucleotide moiety, 3'-2'-in
  • modified 5'-cap structures are regarded as at least one modification comprised in the optimized nucleic acid molecule, preferably in an RNA molecule, according to the present invention.
  • Particularly preferred modified 5'-cap structures are CAP1 (methylation of the ribose of the adjacent nucleotide of m7G), cap2 (additional methylation of the ribose of the 2 nd nucleotide downstream of the m7G), cap3 (additional methylation of the ribose of the 3 rd nucleotide downstream of the m7G), cap4 (additional methylation of the ribose of the 4 lh nucleotide downstream of the m7G), ARCA (anti-reverse cap analogue, modified ARCA (e.g.
  • the methods of making the optimized nucleic acid molecule) of the invention comprise at least the following key step (2): (2) Combining of at least two nucleic acid modules (moieties), to form a combined nucleic acid molecule.
  • the combined nucleic acid molecule is preferably a chimeric molecule.
  • At least one of these moieties encodes a protein or polypeptide of interest.
  • the combined nucleic acid molecule comprises the two moieties in functional relationship to each other.
  • a further moiety encoding a polypeptide or protein of interest is combined with said protein or polypeptide of interest, then the combination occurs such that the combined nucleic acid molecule encodes the two protein elements or polypeptide elements in functional relationship to each other, e.g. as fusion protein.
  • a non- coding moiety i.e.
  • the combi ned nucleic acid molecule encodes protein or polypeptide of interest in functional relationship to the non- coding moiety, e.g. such that the non-coding moiety beneficially influences translation of the protein or polypeptide of interest, or beneficially influences any other functional property, such as RNA stability.
  • Methods for making combined nucleic acid molecules are well established in the art, e.g. Current Protocols in Molecular Biology, Ausubel et al. (ed.), 2003, John Wiley & Sons, Inc., and can be used in step (2). Multiple same or different steps (2) can be performed, either sequentially, or simultaneously.
  • Step (3) relates to a chemical modification as described herein.
  • chemical modification is introduced into a nucleic acid molecule by incorporating a chemically modiefied building block at the stage of synthesizing ⁇ in vivo or in vitro) the respective nucleic acid molecule.
  • Step (3) may be characterized as follows:
  • the step of substituting is characterized in that one building block of the nucleic acid molecule is replaced by a different bui lding block, prefereably selected from the following:
  • the step of adding is characterized in that a further building block is added to the nucleic acid molecule, prefereably selected from the following:
  • lipid bui lding block is added to the nucleic acid molecule
  • the 5'-cap according to (i i-b) can also be introduced post-transcriptionally (e.g., after RNA in vitro transcription using viral or eukaryotic capping enzymes).
  • a substitution can consist of substitution by a naturally occurring bui lding block; respective embodiments are realized as G/C modification, as described below, or as codon optimization, as described below.
  • such substitution can consist of the introduction of a chemical modification (sugar modification, backbone modification, base modification, lipid modification, and introduction of a 5'-cap, al l as described below. Methods for making such substitutions are well established in the art, e.g. Current Protocols in Molecular Biology, Ausubel et al. (ed.), 2003, John Wiley & Sons, Inc.), and can be used in step (3). Multiple same or different steps (3) can be performed, either sequentially, or simultaneously.
  • the steps (2) and (3) are performed simultaneously.
  • step (2) is preferably performed first.
  • step (2) is preceded by the following step (1 ):
  • the method of designing is carried out as fol lows:
  • step (1 ) enables the making of nucleic acid molecules having any combination of desired properties, or the indirect making of polypeptides or proteins (e.g. fusion proteins) having any combinations having any combination of desired properties.
  • any functional property of a nucleic acid molecule or of a polypeptide may be, under some circumstances, a desired property.
  • Functional properties associated with nucleic acid moieties or with polypeptide elements are described throughout the present disclosure. The rational design of step (1 ) enables the targeted combination of any two or more such functional properties.
  • the methods of the invention can also be partially or completely be carried out by a machine or apparatus, based on the guidance provided herein.
  • a respectively suitable apparatus is also comprised by the present invention, in particular, an apparatus suitable for providing an optimized nucleic acid molecule according to the invention.
  • the optimized nucleic acid molecule of the present invention may comprise the following moieties in the following order:
  • ORF stands for one or more open reading frames, each comprised of one or more coding moieties, as described herein.
  • at least one moiety of said ORF encodes a polypeptide or protein of interest (also referred to as coding sequence or cds).
  • the optimized nucleic acid molecule comprises further moieties such as a 5'-cap, a poly(C) sequence and/or an IRES-motif.
  • a 5'-cap may be added, during transcription or post-transcriptionally, to the 5'end of an RNA.
  • nucleic acid molecule of the invention may be modified by a sequence of at least 1 0 cytidines, preferably at least 20 cytidines, more preferably at least 30 cytidines (so-called "poly(C) sequence").
  • the nucleic acid molecule of the invention may contain, especially if the nucleic acid is in the form of an (m)RNA or codes for an mRNA, a poly(C) sequence of typically about 10 to 200 cytidine nucleotides, preferably about 1 0 to 100 cytidine nucleotides, more preferably about 1 0 to 70 cytidine nucleotides or even more preferably about 20 to 50 or even 20 to 30 cytidine nucleotides.
  • the nucleic acid molecule of the invention comprises a poly(C) sequence of 30 cytidine residues.
  • the nucleic acid molecule according to the present invention comprises, preferably in 5'-to-3' direction, at least one 5'- UTR moiety as described above, an ORF, at least one 3'-UTR moiety as described above, a poly(A) sequence or a polyadenylation signal, and a poly(C) sequence or, in 5'-to-3' direction, optional ly a further 5'-UTR, an ORF, at least one 3'-UTR moiety as described above, a poly(A) sequence or a polyadenylation signal, and a poly(C) sequence, or, in 5'-to-3' direction, at least one 5'-UTR moiety as described above, an ORF, optionally a further 3'-UTR, a poly(A) sequence or a polyadenylation signal, and a poly(C) sequence.
  • the present invention also provides an optimized nucleic acid molecule obtainable by a method for generating an optimized nucleic acid molecule according to the present invention as described herei n.
  • nucleic acid of the present invention is useful e.g. in the context of a vector, of a cel l, of a pharmaceutical composition and of medical methods and uses, as described herein below:
  • the present invention provides a vector comprising the optimized nucleic acid sequence as described herein.
  • the preferred embodiments described above for an optimized nucleic acid molecule according to the present invention also apply for an optimized nucleic acid molecule according to the present invention, which is comprised by a vector according to the present invention.
  • the at least one 3'-UTR moiety and/or the at least one 5'-UTR moiety and the ORF are as described above for the optimized nucleic acid molecule according to the present invention, including the preferred embodiments.
  • the vector suitably comprises a cloning site.
  • the cloning site may be any sequence that is suitable for introducing an open reading frame or a sequence comprising an open reading frame, such as one or more restriction sites.
  • the vector comprising a cloning site is preferably suitable for inserting an open reading frame into the vector, preferably for inserting an open reading frame 3' to the 5'-UTR moiety and/or 5' to the 3'-UTR moiety.
  • the cloning site or the ORF is located 3' to the 5'-UTR moiety and/or 5' to the 3'-UTR moiety, preferably in close proximity to the 3'-end of the 5'-UTR moiety and/or to the 5'-end of the 3'-UTR moiety.
  • the cloning site or the ORF may be directly connected to the 3'-end of the 5'-UTR moiety and/or to the 5'-end of the 3'-UTR moiety or they may be connected via a stretch of nucleotides, such as by a stretch of 2, 4, 6, 8, 1 0, 20 etc. nucleotides as described above for the optimized nucleic acid molecule according to the present invention.
  • the vector according to the present invention is suitable for producing the optimized nucleic acid molecule according to the present invention, preferably for producing an RNA, particularly an mRNA according to the present invention, for example, by optionally inserting an open reading frame or a sequence comprising an open reading frame into the vector and transcribing the vector.
  • the vector comprises moieties needed for transcription, such as a promoter, e.g. an RNA polymerase promoter.
  • the vector is suitable for transcription using eukaryotic, prokaryotic, viral or phage transcription systems, such as eukaryotic cells, prokaryotic cells, or eukaryotic, prokaryotic, viral or phage in vitro transcription systems.
  • the vector may comprise a promoter sequence, which is recognized by a polymerase, such as by an RNA polymerase, e.g. by a eukaryotic, prokaryotic, viral, or phage RNA polymerase.
  • a polymerase such as by an RNA polymerase, e.g. by a eukaryotic, prokaryotic, viral, or phage RNA polymerase.
  • the vector comprises a phage RNA polymerase promoter such as an SP6, T3 or T7, preferably a T7 promoter.
  • the vector is suitable for in vitro transcription using a phageenzyme based in vitro transcription system, such as a T7 RNA polymerase based in vitro transcription system.
  • the vector may be used directly for expression of the encoded peptide or protein in cel ls or tissue.
  • the vector comprises particular moieties, which are necessary for expression in those cells/tissue e.g. particular promoter sequences, such as a CMV promoter.
  • the vector may further comprise a poly(A) sequence and/or a polyadenylation signal as described above for the optimized nucleic acid molecule according to the present invention.
  • the vector may be an RNA vector or a DNA vector.
  • the vector is a DNA vector.
  • the vector may be any vector known to the skilled person, such as a viral vector or a plasmid vector.
  • the vector is a plasmid vector, preferably a DNA plasmid vector.
  • an RNA vector according to the present invention comprises a sequence selected from the group consisting of the sequences according to RNA sequences corresponding to DNA sequences described above in relation to the DNA vector according to the present invention.
  • the vector is a circular molecule.
  • the vector is a double-stranded molecule, such as a double-stranded DNA molecule.
  • Such circular, preferably double stranded DNA molecule may be used conveniently as a storage form for the inventive optimized nucleic acid molecule.
  • it may be used for transfection of cells, for example, cultured cells. Also it may be used for in vitro transcription for obtaining an artificial RNA molecule according to the invention.
  • the vector preferably the circular vector, is linearizable, for example, by restriction enzyme digestion.
  • the vector comprises a cleavage site, such as a restriction site, preferably a unique cleavage site, located immediately 3' to the ORF, or - if present - located immediately 3' to the 3'-UTR moiety, or - if present - located 3' to the poly(A) sequence or polyadenylation signal, or - if present - located 3' to the poly(C) sequence, or - if present - located 3' to the histone stem-loop.
  • a cleavage site such as a restriction site, preferably a unique cleavage site, located immediately 3' to the ORF, or - if present - located immediately 3' to the 3'-UTR moiety, or - if present - located 3' to the poly(A) sequence or polyadenylation signal, or - if present - located 3' to the poly(C) sequence, or
  • the product obtained by l inearizing the vector terminates at the 3'end with the 3'-end of the ORF, or - if present - with the 3'-end of the 3'-UTR moiety, or - if present - with the 3'-end of the poly(A) sequence or polyadenylation signal, or - if present - with the 3'-end of the poly(C) sequence.
  • a restriction site preferably a unique restriction site, is preferably located immediately 3' to the 3'-end of the optimized nucleic acid molecule.
  • the present invention relates to a cell comprising the optimized nucleic acid molecule according to the present invention or the vector according to the present invention.
  • the cell may be any cell, such as a bacterial cell, insect cell, plant cell, vertebrate cell, e.g. a mammalian cell. Such cell may be, e.g., used for replication of the vector of the present invention, for example, in a bacterial cell .
  • the cell may be used for transcribing the optimized nucleic acid molecule or the vector according to the present invention and/or translating the open reading frame of the optimized nucleic acid molecule or the vector according to the present invention.
  • the cell may be used for recombinant protein production.
  • the cells according to the present invention are, for example, obtainable by standard nucleic acid transfer methods, such as standard transfection, transduction or transformation methods.
  • the optimized nucleic acid molecule or the vector according to the present invention may be transferred into the cell by electroporation, lipofection, e.g. based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or based on cationic polymers, such as DEAE-dextran or polyethylenimine etc.
  • the cell is a mammalian cell, such as a cell of human subject, a domestic animal, a laboratory animal, such as a mouse or rat cel l.
  • Cells include in particular cell lines, primary cells, cells in tissue or subjects.
  • cell types allowing cell culture may be suitable for the present invention.
  • the cell may be a cell of an established cell line, such as a CHO, BHK, 293T, COS-7, HeLa, HEPG2 and HEK, etc. or the cell may be a primary cell, such as a human dermal fibroblast (HDF) cell etc., preferably a cell isolated from an organism.
  • HDF human dermal fibroblast
  • the cell is an isolated cell of a mammalian subject, preferably of a human subject.
  • the cell may be an immune cell, such as a dendritic cell, a cancer or tumor cell, or any somatic cell etc., preferably of a mammalian subject, preferably of a human subject.
  • the present invention provides a pharmaceutical composition comprising the optimized nucleic acid molecule according to the present invention, the vector according the present invention, or the cell according to the present invention.
  • the pharmaceutical composition according to the invention may be used, e.g., as a vaccine, for example, for genetic vaccination.
  • the ORF may, e.g., encode an antigen to be administered to a patient for vaccination.
  • the pharmaceutical composition according to the present invention is a vaccine.
  • the pharmaceutical composition according to the present invention may be used, e.g., for gene therapy.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable vehicles, diluents and/or excipients and/or one or more adjuvants.
  • a pharmaceutically acceptable vehicle typically includes a liquid or non-liquid basis for the inventive pharmaceutical composition.
  • the pharmaceutical composition is provided in liquid form.
  • the vehicle is based on water, such as pyrogen-free water, isotonic saline or buffered (aqueous) solutions, e.g phosphate, citrate etc. buffered solutions.
  • the buffer may be hypertonic, isotonic or hypotonic with reference to the specific reference medium, i.e.
  • the buffer may have a higher, identical or lower salt content with reference to the specific reference medium, wherein preferably such concentrations of the afore mentioned salts may be used, which do not lead to damage of mammalian cells due to osmosis or other concentration effects.
  • Reference media are e.g. liquids occurring in "in vivo" methods, such as blood, lymph, cytosolic liquids, or other body liquids, or e.g. liquids, which may be used as reference media in “in vitro” methods, such as common buffers or liquids. Such common buffers or liquids are known to a skilled person. Ringer-Lactate solution is particularly preferred as a liquid basis.
  • compatible solid or liquid fillers or diluents or encapsulating compounds suitable for administration to a patient may be used as well for the inventive pharmaceutical composition.
  • the term "compatible” as used herein preferably means that these components of the inventive pharmaceutical composition are capable of being mixed with the inventive optimized nucleic acid, vector or cells as defined herein in such a manner that no interaction occurs which would substantially reduce the pharmaceutical effectiveness of the inventive pharmaceutical composition under typical use conditions.
  • the pharmaceutical composition according to the present invention may optional ly further comprise one or more additional pharmaceutically active components.
  • a pharmaceutically active component in this context is a compound that exhibits a therapeutic effect to heal, ameliorate or prevent a particular indication or disease.
  • Such compounds include, without implying any limitation, peptides or proteins, nucleic acids, (therapeutical ly active) low molecular weight organic or inorganic compounds (molecular weight less than 5000, preferably less than 1 000), sugars, antigens or antibodies, therapeutic agents already known in the prior art, antigenic cells, antigenic cellular fragments, cellular fractions, cell wall components (e.g. polysaccharides), modified, attenuated or de-activated (e.g. chemically or by irradiation) pathogens (virus, bacteria etc.).
  • the pharmaceutical composition according to the invention may comprise a carrier for the optimized nucleic acid molecule or the vector.
  • Such a carrier may be suitable for mediating dissolution in physiological acceptable liquids, transport and cellular uptake of the pharmaceutical active optimized nucleic acid molecule or the vector.
  • a carrier may be a component which may be suitable for depot and delivery of an optimized nucleic acid molecule or vector according to the invention.
  • Such components may be, for example, cationic or polycationic carriers or compounds which may serve as transfection or complexation agent.
  • transfection or complexation agents are cationic or polycationic compounds, including protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), poly-arginine, basic polypeptides, cel l penetrating peptides (CPPs), including HIV-binding peptides, HIV-1 Tat (HIV), Tat- derived peptides, Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex), MAP, ALA or protein transduction domains (PTDs), PpT620, proline-rich peptides, arginine- rich peptides, lysine-rich peptides, MPG-peptide(s), Pep-1 , L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides (particularly from Drosophila antennapedia), pA
  • PLL poly
  • cationic or polycationic compounds may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • cationic polysaccharides for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • PEI polyethyleneimine
  • DOTMA N [1 -(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidi ne, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: ⁇ , ⁇ - ditetradecanoyl-N-(-trimethylammonioacetyl)diethanolamine chloride, CL1P1 : rac-[(2,3- dioctadecyloxypropyl)(2-hydroxyethyl
  • modified polyaminoacids such as alpha-aminoacid- polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N-ethyl-4- vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
  • modified Amidoamines such as pAMAM (poly(amidoamine)), etc.
  • modified polybetaaminoester (PBAE) such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1 -pentanol polymers, etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
  • polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), etc.
  • polyallylamine sugar backbone based polymers
  • preferred cationic or polycationic proteins or peptides which can be used as an adjuvant by complexing the optimized nucleic acid molecule or the vector, preferably an RNA, of the composition, may be selected from followi ng proteins or peptides having the following total formula (VII): (Arg)i;(Lys)m;(His)n;(0m) 0 ;(Xaa)!
  • l + m + n +o + x 8- 1 5
  • I, m, n or o independently of each other may be any number selected from 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 1 1 , 12, 1 3, 14 or 1 5, provided that the overal l content of Arg, Lys, His and Orn represents at least 50% of all amino acids of the oligopeptide
  • x may be any number selected from 0, 1 , 2, 3 or 4, provided, that the overall content of Xaa does not exceed 50 % of all amino acids of the oligopeptide.
  • oligoarginines in this context are e.g. Arg7, Arg8, Arg9, Arg7, H3R9, R9H3, H3R9H3, YSSR9SSY, (RKH)4, Y(RKH)2R, etc.
  • cationic or polycationic compounds or carriers may be cationic or polycationic peptides or proteins, which preferably comprise or are additionally modified to comprise at least one -SH moiety.
  • a cationic or polycationic carrier is selected from cationic peptides having the following sum formula (VII):
  • the cationic or polycationic peptide or protein when defined according to formula ⁇ (Arg)i;(Lys) m ;(His) n ;(Orn)o;(Xaa)x ⁇ (formula (VII)) as shown above and which comprise or are additionally modified to comprise at least one -SH moeity, may be, without being restricted thereto, selected from subformula (Vila):
  • Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids are disclosed in WO201 1/026641 . Such conjugates are also encompassed by the present invention.
  • the polypeptide or protein element of the present invention may be selected to allow preparation of a disulfide-linked polyethyleneglycol/peptide conjugate.
  • the polymeric carrier which may be used to complex the optimized nucleic acid molecule or the vector may be selected from a polymeric carrier molecule according to generic formula (IV):
  • PI and P3 are different or identical to each other and represent a linear or branched hydrophilic polymer chain, each P1 and P3 exhibiting at least one -SH group, capable to form a disulfide linkage upon condensation with component P2, or alternatively with (AA), (AA)x, or [(AA)x]z if such components are used as a linker between P1 and P2 or P3 and P2) and/or with further components (e.g.
  • the linear or branched hydrophilic polymer chain selected independent from each other from polyethylene glycol (PEG), poly-N- (2-hydroxypropyl)methacrylamide, poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hyclroxyalkyl L-asparagine), poly(2- (methacryloyloxy)ethyl phosphorylcholine), hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the hydrophilic polymer chain exhibits a molecular weight of about 1 kDa to about 1 00 kDa, preferably of about 2 kDa to about 25 kDa; or more preferably of about 2 kDa to about 1 0 kDa, e.g. about 5 kDa to about 25 kDa or 5 kDa to about 1 0 kDa;
  • a cationic or polycationic peptide or protein e.g. as defined above for the polymeric carrier formed by disulfide-crosslinked cationic components, and preferably having a length of about 3 to about 100 amino acids, more preferably havi ng a length of about 3 to about 50 ami no acids, even more preferably having a length of about 3 to about 25 amino acids, e.g. a length of about 3 to 10, 5 to 1 5, 1 0 to 20 or 1 5 to 25 amino acids, more preferably a length of about 5 to about 20 and even more preferably a length of about 10 to about 20; or is a cationic or polycationic polymer, e.g.
  • the polymeric carrier formed by disulfide-crosslinked cationic components typically having a molecular weight of about 0.5 kDa to about 30 kDa, including a molecular weight of about 1 kDa to about 20 kDa, even more preferably of about 1 .5 kDa to about 1 0 kDa, or having a molecular weight of about 0.5 kDa to about 1 00 kDa, including a molecular weight of about 10 kDa to about 50 kDa, even more preferably of about 10 kDa to about 30 kDa; each P2 exhibiting at least two -SH- moieties, capable to form a disulfide linkage upon condensation with further components P2 or component(s) P1 and/or P3 or alternatively with further components (e.g. (AA), (AA)x, or [(AA)x]z);
  • further components e.g. (AA), (AA)x, or [(AA)x]z
  • n S preferably represents sulphur or a -SH carrying moiety, which has formed a (reversible) disulfide bond.
  • the (reversible) disulfide bond is preferably formed by condensation of -SH-moieties of either components PI and P2, P2 and P2, or P2 and P3, or optional ly of further components as defined herein (e.g. L, (AA), (AA)x, [(AA)x]z, etc);
  • the -SH group may be part of the structure of these components or added by a modification as defined below;
  • ligand is an optional ligand, which may be present or not, and may be selected independent from the other from RGD, Transferrin, Folate, a signal peptide or signal sequence, a localization signal or sequence, a nuclear localization signal or sequence (NLS), an antibody, a cell penetrating peptide, (e.g. TAT or KALA), a ligand of a receptor (e.g. cytokines, hormones, growth factors etc), small molecules (e.g. carbohydrates like mannose or galactose or synthetic ligands), small molecule agonists, inhibitors or antagonists of receptors (e.g. RGD peptidomimetic analogues), or any further protein as defined herein, etc.;
  • a range of about 1 to 50 is an integer, typically selected from a range of about 1 to 50, preferably from a range of about 1 , 2 or 3 to 30, more preferably from a range of about 1 , 2, 3, 4, or 5 to 25, or a range of about 1 , 2, 3, 4, or 5 to 20, or a range of about 1 , 2, 3, 4, or 5 to 1 5, or a range of about 1 , 2, 3, 4, or 5 to 10, including e.g. a range of about 4 to 9, 4 to 1 0, 3 to 20, 4 to 20, 5 to 20, or 1 0 to 20, or a range of about 3 to 1 5, 4 to 1 5, 5 to 1 5, or 1 0 to 1 5, or a range of about 6 to 1 1 or 7 to 1 0.
  • n is in a range of about 1 , 2, 3, 4, or 5 to 1 0, more preferably in a range of about 1 , 2, 3, or 4 to 9, in a range of about 1 , 2, 3, or 4 to 8, or in a range of about 1 , 2, or 3 to 7.
  • Each of hydrophilic polymers P1 and P3 typically exhibits at least one -SH group, wherein the at least one -SH group is capable to form a disulfide linkage upon reaction with component P2 or with component (AA) or (AA) X , if used as linker between P1 and P2 or P3 and P2 as defined below and optionally with a further component, e.g. L and/or (AA) or (AA)x, e.g. if two or more -SH-moieties are contained.
  • a further component e.g. L and/or (AA) or (AA)x, e.g. if two or more -SH-moieties are contained.
  • the term "-S-S-" in these formulae may also be written as “-S-Cys", as “-Cys-S” or as “-Cys- Cys-”.
  • the term “-Cys-Cys-” does not represent a peptide bond but a linkage of two cysteines via their -SH groups to form a disulfide bond.
  • the term “-Cys- Cys-” also may be understood generally as “-(Cys-S)-(S-Cys)-", wherein in this specific case S indicates the sulphur of the -SH group of cysteine.
  • the terms "-S-Cys” and “-Cys-S” indicate a disulfide bond between a -SH containing moiety and a cysteine, which may also be written as “-S-(S-Cys)" and "-(Cys-S)-S".
  • the hydrophilic polymers P1 and P3 may be modified with a -SH moiety, preferably via a chemical reaction with a compound carrying a -SH moiety, such that each of the hydrophi lic polymers PI and P3 carries at least one such -SH moiety.
  • a compound carrying a -SH moiety may be e.g.
  • Such a compound may also be any non-amino compound or moiety, which contains or al lows to introduce a - SH moiety into hydrophilic polymers P1 and P3 as defined herein.
  • Such non-amino compounds may be attached to the hydrophi lic polymers P1 and P3 of formula (IV) of the polymeric carrier via chemical reactions or binding of compounds, e.g. by binding of a 3-thio propionic acid or thioimolane, by amide formation (e.g.
  • alkenes or alkines e.g., alkenes or alkines
  • imine or hydrozone formation aldehydes or ketons, hydrazins, hydroxylamins, amines
  • complexation reactions avidin, biotin, protein G
  • Sn- type substitution reactions e.g halogenalkans, thiols, alcohols, amines, hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts
  • a particularly preferred PEG derivate in this context is alpha-Methoxy-omega-mercapto poly(ethylene glycol).
  • the SH group e.g.
  • each of hydrophilic polymers PI and P3 typically exhibits at least one -SH- group preferably at one terminal end, but may also contain two or even more -SH groups, which may be used to additionally attach further components as defined herein, preferably further functional peptides or proteins e.g. a ligand, an amino acid component (AA) or (AA)x, antibodies, cell penetrating peptides or enhancer peptides (e.g. TAT, KALA), etc.
  • the polymeric carrier molecule can additionally contain an amino acid component (AA) X , wherein x is an integer selected from a range of about 1 to 1 00.
  • the amino acid component (AA) * can comprise an aromatic amino acid component, a hydrophil ic amino acid component, a lipophi lic amino acid component, a weak basic amino acid component, a signal peptide, localization signal or sequence, a nuclear localization signal or sequence, a cell penetrating peptide, a therapeutically active protein or peptide, an antigen or an antigenic epitope, a tumour antigen, a pathogenic antigen (an animal antigen, a viral antigen, a protozoal antigen, a bacterial antigen, an allergic antigen), an autoimmune antigen, or a further antigen, an allergen, an antibody, an immunostimulatory protein or peptide, an antigen-specific T-cell receptor, or another protein or peptide suitable for a specific (therapeutic) application.
  • n is as defined before, preferably in a range of about 1 , 2, 3, 4, or 5 to 10;
  • a is an integer, selected independent from integer b from a range of about 1 to 50, preferably in a range of about 1 , 2, 3, 4, or 5 to 10, and
  • b is an integer, selected independent from integer a from a range of about 1 to 50, preferably in a range of about 1 , 2, 3, 4, or 5 to 10,
  • Component P 2 can be a cationic or polycationic peptide selected from protamine, nucleoline, spermine or spermidine, poly-L-lysine (PLL), basic polypeptides, poly-arginine, cell penetrating peptides (CPPs), chimeric CPPs, Transportan, or MPG peptides, HIV-binding peptides, Tat, HIV-1 Tat (HIV), Tat-derived peptides, oligoarginines, members of the penetratin family, Penetratin, Antennapedia-derived peptides (particularly from Drosophila antennapedia), pAntp, plsl, etc., antimicrobial-derived CPPs, Buforin-2, Bac71 5-24
  • Such polymeric carrier molecules can be incorporated in a polymeric carrier cargo complex, wherein the polymeric carrier cargo is formed of said polymeric carrier molecule and a nucleic acid.
  • Said nucleic acid can be provided in a molar ratio of about 5 to 10000 of polymeric carrier molecule : nucleic acid.
  • the polymeric cargo complexes enable expression of a therapeutically active protein or peptide, an antigen, including tumor antigens, pathogenic antigens, animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens, autoimmune antigens, allergens, antibodies, immunostimulatory proteins or peptides, or antigen-specific T-cell receptors.
  • an antigen including tumor antigens, pathogenic antigens, animal antigens, viral antigens, protozoal antigens, bacterial antigens, allergic antigens, autoimmune antigens, allergens, antibodies, immunostimulatory proteins or peptides, or antigen-specific T-cell receptors.
  • the polymeric carrier molecule can be prepared by a method comprising fol lowing steps: a) providing at least one cationic or polycationic protein or peptide as component P 2 as defined herein and/or at least one cationic or polycationic polymer as component P 2 as defined according to one of claims 1 to 8, and optionally at least one further component (AA) X , mixing these components, preferably in a basic milieu, preferably in the presence of oxygen or a further starter which leads to mi ld oxidation conditions, and thereby condensing and thus polymerizing these components with each other via disulfide bonds in a polymerization condensation or polycondensation to obtain a repetitive component H-[S-P -S] n -H or H ⁇ [S-P 2 -S] a [S-(AA) x -S] b ⁇ H;
  • step b) providing a hydrophilic polymer P 1 and/or P 3 as defined according to any of claims 1 to 8, optionally modified with a ligand L and/or an amino acid component (AA) X as defined according to any of claims 1 to 8; c) mixing the hydrophilic polymer P 1 and/or P 3 according to step b) with the repetitive component H-[S-P 2 -S] sanction-H or H ⁇ [S-P -S] a [S-(AA) x -S]b ⁇ H obtai ned according to step a) in a ratio of about 2 : 1 , and thereby typically terminating the polymerization condensation or polycondensation reaction and obtaining the inventive polmeric carrier molecule according to formula (IV) or (IVa); d) optionally purifyi ng the polymeric carrier molecule obtained according to step c);
  • step c) optionally adding a nucleic acid as defined herein to the polymeric carrier obtained according to step c) or d) and complexing the nucleic acid with the polymeric carrier obtained according to step c) or d) to obtain a polymeric carrier cargo complex as defined according to any of claims 9 to 1 2.
  • cationic or polycationic compounds which can be used as transfection or complexation agent may include cationic polysaccharides, for example chitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI), cationic lipids, e.g.
  • PEI polyethyleneimine
  • DOTMA [1 -(2,3- sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethyl hydroxyethyl ammonium bromide, DOTAP: dioleoyloxy-3- (trimethylammonio)propane, DC-6-14: 0,0-ditetradecanoyl-N-(ct- trimethylammonioacetyl)diethanolamine chloride, CLIP1 : rac-[(2,3-dioctadecyloxypropyl)(2- hydroxyethyl)]-dimethyl
  • modified polyaminoacids such as ⁇ -aminoacid-polymers or reversed polyamides, etc.
  • modified polyethylenes such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), etc.
  • modified acrylates such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.
  • modified Amidoamines such as pAMAM (poly(amidoamine)), etc.
  • modified polybetaaminoester (PBAE) such as diamine end modified 1 ,4 butanediol diacrylate-co-5-amino-1 -pentanol polymers, etc.
  • dendrimers such as polypropylamine dendrimers or pAMAM based dendrimers, etc.
  • polyimine(s) such as PEI: poly(ethyleneimine), poly(propyleneimine), etc.
  • polyallylamine sugar backbone
  • the pharmaceutical composition according to the invention may comprise an adjuvant in order to enhance the immunostimulatory properties of the pharmaceutical composition.
  • an adjuvant may be understood as any compound, which is suitable to support administration and delivery of the components such as the optimized nucleic acid molecule or vector comprised in the pharmaceutical composition according to the i nvention.
  • an adjuvant may, without being bound thereto, initiate or increase an immune response of the innate immune system, i.e. a non-specific immune response.
  • the pharmaceutical composition according to the invention typically initiates an adaptive immune response directed to the antigen encoded by the optimized nucleic acid molecule.
  • the pharmaceutical composition according to the invention may generate an (supportive) innate immune response due to addition of an adjuvant as defined herein to the pharmaceutical composition according to the invention.
  • Such an adjuvant may be selected from any adjuvant known to a skilled person and suitable for the present case, i.e. supporting the induction of an immune response in a mammal.
  • the adjuvant may be selected from the group consisting of, without being limited thereto, TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminium hydroxide, ADJ UMERTM (polyphosphazene); aluminium phosphate gel; glucans from algae; algammulin; aluminium hydroxide gel (alum); highly protein-adsorbing aluminium hydroxide gel; low viscosity aluminium hydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%), Pluronic L121 (1 .25%), phosphate-buffered saline, pH 7.4); AVRIDINETM (propanediamine); BAY R1005TM ((N-(2-deoxy-2-L-leucyla)
  • Suitable adjuvants may also be selected from cationic or polycationic compounds wherein the adjuvant is preferably prepared upon complexing the optimized nucleic acid molecule or the vector of the pharmaceutical composition with the cationic or polycationic compound. Association or complexing the optimized nucleic acid molecule or the vector of the pharmaceutical composition with cationic or polycationic compounds as defined herein preferably provides adjuvant properties and confers a stabilizing effect to the optimized nucleic acid molecule or the vector of the pharmaceutical composition.
  • the ratio of nucleic acid (the optimized nucleic acid or vector comprising the same) to cationic or polycationic compound may be calculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) of the entire nucleic acid complex.
  • N/P-ratio nitrogen/phosphate ratio
  • 1 ⁇ g RN A typically contains about 3 nmol phosphate residues, provided the RNA exhibits a statistical distribution of bases.
  • 1 pg peptide typical ly contains about x nmol nitrogen residues, dependent on the molecular weight and the number of basic amino acids.
  • protamine molecular weight about 4250 g mol, 21 nitrogen atoms, when protamine from salmon is used
  • N/P ratio of about 0.81 can be calculated.
  • mass ratio of about 8: 1 RNA/protamine an N/P ratio of about 0.2 can be calculated.
  • an N/P-ratio is preferably in the range of about 0.1 -1 0, preferably in a range of about 0.3-4 and most preferably in a range of about 0.5-2 or 0.7-2 regarding the ratio of nucleic acid:peptide in the complex, and most preferably in the range of about 0.7-1 .5.
  • Patent application W0201 0/037539 describes an immunostimulatory composition and methods for the preparation of an immunostimulatory composition. Accordingly, in a preferred embodiment of the invention, the composition is obtained in two separate steps in order to obtain both, an efficient immunostimulatory effect and efficient translation of the optimized nucleic acid molecule according to the invention.
  • a so called “adjuvant component” is prepared by complexing - in a first step - the optimized nucleic acid molecule or vector, preferably an RNA, of the adjuvant component with a cationic or polycationic compound in a specific ratio to form a stable complex.
  • the ratio of the nucleic acid and the cationic or polycationic compound in the adjuvant component is typically selected in a range that the nucleic acid is entirely complexed and no free cationic or polycationic compound or only a small amount remains in the composition.
  • the ratio of the adjuvant component i.e. the ratio of the nucleic acid to the cationic or polycationic compound is selected from a range of about 6:1 (w/w) to about 0,25:1 (w/w), more preferably from about 5:1 (w/w) to about 0,5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1 :1 (w/w) or of about 3 :1 (w/w) to about 1 :1 (w/w), and most preferably a ratio of about 3: 1 (w/w) to about 2:1 (w/w).
  • the optimized nucleic acid molecule or vector, preferably an RNA molecule, according to the invention is added in a second step to the complexed nucleic acid molecule, preferably an RNA, of the adjuvant component in order to form the (immunostimulatory) composition of the invention.
  • the artificial acid molecule or vector, preferably an RNA, of the invention is added as free nucleic acid, i.e. nucleic acid, which is not complexed by other compounds.
  • the free optimized nucleic acid molecule or vector is not complexed and will preferably not undergo any detectable or significant complexation reaction upon the addition of the adjuvant component.
  • the pharmaceutical composition according to the present invention preferably comprises a "safe and effective amount" of the components of the pharmaceutical composition, particularly of the inventive optimized nucleic acid molecule, the vector and/or the cel ls as defined herein.
  • a "safe and effective amount” means an amount sufficient to significantly i nduce a positive modification of a disease or disorder as defined herein.
  • a "safe and effective amount” preferably avoids serious side-effects and permits a sensible relationship between advantage and risk. The determination of these limits typically lies within the scope of sensible medical judgment.
  • kits or kit of parts comprising an optimized nucleic acid molecule according to the invention, a vector according to the invention, a cell according to the invention, and/or a pharmaceutical composition according to the invention.
  • kit or kits of parts may, additionally, comprise instructions for use, cel ls for transfection, an adjuvant, a means for administration of the pharmaceutical composition, a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable solution for dissolution or dilution of the optimized nucleic acid molecule, the vector, the cel ls or the pharmaceutical composition.
  • the optimized nucleic acid molecules of the present invention are suitable for in vivo administration to humans and animals, particularly in medical methods.
  • Gene therapy and genetic vaccination belong to the most promising and quickly developing medical methods of our modern times. They may provide highly specific and individual options for therapy of a large variety of diseases. Particularly, inherited genetic diseases, infectious diseases, neoplasms (e.g.
  • cancer or tumour diseases autoimmune diseases, inflammatory diseases, diseases of the blood and blood-formi ng organs, endocrine, nutritional and metabolic diseases, diseases of the nervous system, diseases of the circulatory system, diseases of the respiratory system, diseases of the digestive system, diseases of the skin and subcutaneous tissue, diseases of the musculoskeletal system and connective tissue, and diseases of the genitourinary system, independently if they are inherited or acquired, may be the subject of such treatment approaches. Also, it is envisaged to prevent early onset of such diseases by these approaches.
  • the present invention provides the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention for use as a medicament, for example, as vaccine (in genetic vaccination) or in gene therapy.
  • the use can comprise the administration of the optimized nucleic acid molecule according to the present invention, the vector accordi ng to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention to a patient in need thereof.
  • the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cel l according to the present invention, or the pharmaceutical composition according to the present invention are particularly suitable for any medical appl ication which makes use of the therapeutic action or effect of peptides, polypeptides or proteins, or where supplementation of a particular peptide or protein is needed or beneficial.
  • the present invention provides the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention for use in the treatment or prevention of diseases or disorders amenable to treatment by the therapeutic action or effect of peptides, polypeptides or proteins or amenable to treatment by supplementation of a particular peptide, polypeptide or protein.
  • the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may be used for the treatment or prevention of genetic diseases, autoimmune diseases, cancerous or tumour-related diseases, infectious diseases, chronic diseases or the like, e.g., by genetic vaccination or gene therapy.
  • such therapeutic treatments which benefit from an increased and prolonged presence of therapeutic peptides, polypeptides or proteins or from more immunogenic properties of the therapeutic peptides, polypeptides or proteins in a subject to be treated are especially suitable as medical application in the context of the present invention.
  • a particularly suitable medical application for the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention is vaccination.
  • the present invention provides the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention for vaccination of a subject, preferably a mammalian subject, more preferably a human subject.
  • Preferred vaccination treatments are vaccination agai nst infectious diseases, such as bacterial, protozoal or viral infections, and anti-tumour- vaccination. Such vaccination treatments may be prophylactic or therapeutic.
  • the protein of interest encoded by the optimized nucleic acid molecule may be selected.
  • the open reading frame may code for a protein that has to be supplied to a patient suffering from total lack or at least partial loss of function of a protein, such as a patient suffering from a genetic disease.
  • the open reading frame may be chosen from an ORF coding for a peptide or protein which beneficial ly influences a disease or the condition of a subject.
  • the open reading frame may code for a peptide or protein which effects down-regulation of a pathological overproduction of a natural peptide or protein or elimination of cells expressing pathologically a protein or peptide. Such lack, loss of function or overproduction may, e.g., occur in the context of tumour and neoplasia, autoimmune diseases, allergies, infections, chronic diseases or the like.
  • the open reading frame may code for an antigen or immunogen, e.g. for an epitope of a pathogen or for a tumour antigen.
  • the optimized nucleic acid molecule or the vector according to the present invention comprises an ORF encoding an amino acid sequence comprising or consisting of an antigen or immunogen, e.g. an epitope of a pathogen or a tumour-associated antigen, a 3'-UTR moiety as described above and/or a 5'-UTR moiety as described above, and optional further components, such as a poly(A) sequence etc.
  • the optimized nucleic acid molecule according to the present invention is RNA, preferably mRNA, since DNA harbours the risk of eliciting an anti-DNA immune response and tends to insert into genomic DNA.
  • a viral delivery vehicle such as an adenoviral delivery vehicle
  • the optimized nucleic acid molecule or the vector is a DNA molecule.
  • Pathologically altered gene expression may result in lack or overproduction of essential gene products, for example, signalling factors such as hormones, housekeeping factors, metabolic enzymes, structural proteins or the like. Altered gene expression may not only be due to mis-regulation of transcription and/or translation, but also due to mutations within the ORF coding for a particular protein. Pathological mutations may be caused by e.g. chromosomal aberration, or by more specific mutations, such as point or frame-shift- mutations, all of them resulting in limited functionality and, potentially, total loss of function of the gene product.
  • misregulation of transcription or translation may also occur, if mutations affect genes encoding proteins which are involved in the transcriptional or translational machinery of the cel l. Such mutations may lead to pathological up- or down- regulation of genes which are - as such - functional. Genes encoding gene products which exert such regulating functions, may be, e.g., transcription factors, signal receptors, messenger proteins or the like. However, loss of function of such genes encoding regulatory proteins may, under certain circumstances, be reversed by artificial introduction of other factors acting further downstream of the impaired gene product. Such gene defects may also be compensated by gene therapy via substitution of the affected gene itself.
  • Pathologically altered gene expression may result in lack or overproduction of essential gene products, for example, signal ling factors such as hormones, housekeeping factors, metabolic enzymes, structural proteins or the like. Altered gene expression may not only be due to mis-regulation of transcription and/or translation, but also due to mutations within the ORF coding for a particular protein. Pathological mutations may be caused by e.g. chromosomal aberration, or by more specific mutations, such as point or frame-shift-mutations, all of them resulting in limited functionality and, potentially, total loss of function of the gene product.
  • misregulation of transcription or translation may also occur, if mutations affect genes encoding proteins which are involved in the transcriptional or translational machinery of the cell. Such mutations may lead to pathological up- or down-regulation of genes which are - as such - functional. Genes encoding gene products which exert such regulating functions, may be, e.g., transcription factors, signal receptors, messenger proteins or the like. However, loss of function of such genes encoding regulatory proteins may, under certain circumstances, be reversed by artificial introduction of other factors acting further downstream of the impaired gene product. Such gene defects may also be compensated by gene therapy via substitution of the affected gene itself.
  • Optimized nucleic acid of the present invention can be used as vector for gene therapy.
  • optimized nucleic acid of the present invention can be used to encode any kind of protein suitable for use in molecular therapy.
  • Illustrative examples comprise insulin, EPO and the like.
  • vaccines may be subdivided into “first”, “second” and “third” generation vaccines.
  • First generation vaccines are, typically, whole-organism vaccines. They are based on either live and attenuated or ki lled pathogens, e.g. viruses, bacteria or the like.
  • second generation vaccines are, typically, subunit vaccines, consisting of defined antigens or recombinant protein components which are derived from pathogens.
  • Genetic vaccines i.e. vaccines for genetic vaccination, are usually understood as "third generation” vaccines. They are typically composed of genetically engineered nucleic acid molecules which allow expression of peptide or protein (antigen) fragments characteristic for a pathogen or a tumor antigen in vivo. Genetic vaccines are expressed upon administration to a patient after uptake by target cells. Expression of the administered nucleic acids results in production of the encoded proteins. In the event these proteins are recognized as foreign by the patient's immune system, an immune response is triggered.
  • genetic vaccination or gene therapy are essential ly based on the administration of nucleic acid molecules to a patient and subsequent transcription and/or translation of the encoded genetic information.
  • genetic vaccination or gene therapy may also comprise methods which include isolation of specific body cells from a patient to be treated, subsequent in ex wVo transfection of such cells, and re-administration of the treated cells to the patient. 6.5.3 Route of administration
  • the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may be administered orally, parenterally, by inhalation spray, topical ly, rectally, nasally, buccally, vaginally, via an implanted reservoir or via jet injection.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, intracranial, transdermal, intradermal, intrapulmonal, intraperitoneal, intracardial, intraarterial, and sublingual injection or infusion techniques.
  • the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention is administered via needle- free injection (e.g. jet injection).
  • the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention is administered parenterally, e.g.
  • Steri le injectable forms of the inventive pharmaceutical composition may be aqueous or oleaginous suspension. These suspensions may be formulated accordi ng to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Preferably, the solutions or suspensions are administered via needle-free injection (e.g. jet injection).
  • the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may also be administered oral ly in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions.
  • the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs.
  • the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention may be formulated in a suitable ointment suspended or dissolved in one or more carriers.
  • the use as a medicament comprises the step of transfection of mammalian cells, preferably in vitro or ex vivo transfection of mammalian cells, more preferably in vitro transfection of isolated cells of a subject to be treated by the medicament. If the use comprises the in vitro transfection of isolated cells, the use as a medicament may further comprise the readministration of the transfected cells to the patient.
  • the use of the inventive optimized nucleic acid molecules or the vector as a medicament may further comprise the step of selection of successfully transfected isolated cells. Thus, it may be beneficial if the vector further comprises a selection marker.
  • the use as a medicament may comprise in vitro transfection of isolated cells and purification of an expression-product, i.e. the encoded peptide or protein from these cells. This purified peptide or protein may subsequently be administered to a subject in need thereof.
  • the present invention also provides a method for treating or preventing a disease or disorder as described above comprising administering the optimized nucleic acid molecule according to the present invention, the vector according to the present invention, the cell according to the present invention, or the pharmaceutical composition according to the present invention to a subject in need thereof.
  • the present invention provides a method for treating or preventing a disease or disorder comprising transfection of a cell with an optimized nucleic acid molecule according to the present invention or with the vector according to the present invention. Said transfection may be performed in vitro, ex vivo or in vivo.
  • transfection of a cell is performed in vitro and the transfected cell is administered to a subject in need thereof, preferably to a human patient.
  • the cel l which is to be transfected in vitro is an isolated cell of the subject, preferably of the human patient.
  • the present invention provides a method of treatment comprising the steps of isolating a cell from a subject, preferably from a human patient, transfecting the isolated cel l with the optimized nucleic acid according to the present invention or the vector according to the present invention, and administering the transfected cell to the subject, preferably the human patient.
  • the method of treating or preventing a disorder according to the present invention is preferably a vaccination method or a gene therapy method as described above.
  • Figure 1 shows a western blot to detect HA proteins in cell lysates (A) and cell culture supernatant (B) using an anti HA (H I NT ) protein specific antibody.
  • M protein marker lane; 1 : recombinant HA protein (positive control); 2: ⁇ -SGG-ferritin; 3: ⁇ ," 4: negative control; 5: HAAT -C3d_P28; 6: ⁇ - foldon.
  • the size ladder (kDa) of the protein marker is shown on the left of panel (A). See Example 2.
  • Figure 2 shows IgGI and lgG2a titers of mice immunized with the indicated formulated HA mRNA vaccines. RiLa served as a negative control. Antibody titers were measured at day 21 and day 28. (A) and (B) shows HA-specific IgGI antibody titers; (C) and (D) shows HA-specific lgG2a antibody titers. The horizontal bar indicates the median. Every data poi nt represents one individual specimen. See Example 4.
  • Figure 3 shows HI titers of mice immunized with the indicated formulated HA mRNA vaccines.
  • RiLa served as a negative control.
  • HI titers were determined at day 28 (1 week after boost immunization).
  • HI titers of >40 are associated with a protection from influenza virus infection (indicated by dashed line). Every data point represents one individual specimen. See Example 5.
  • Figure 4 shows lgG1 and lgG2a titers of mice immunized with the indicated formulated HA mRNA vaccines. RiLa served as a negative control.
  • Antibody titers were measured at day 35 and day 49.
  • (A) and (B) shows HA-specific lgG1 antibody titers;
  • (C) and (D) shows HA-specific lgG2a antibody titers.
  • the horizontal bar indicates the median. Every data point represents one individual specimen. See Example 6.
  • Figure 5 shows HI titers of mice immunized with the indicated formulated HA mRNA vaccines.
  • RiLa served as a negative control.
  • HI titers were determined at day 49 (4 weeks after boost immunization).
  • HI titers of >40 are associated with a protection from influenza virus infection (indicated by dashed line). Every data point represents one individual specimen. See Example 7.
  • Figure 6 shows HI titers of mice immunized with the indicated formulated HA mRNA vaccines.
  • RiLa served as a negative control.
  • HI titers were determined at day 14 (2 weeks after boost immunization (pB)).
  • HI titers of >40 are associated with a protection from influenza virus infection (indicated by dashed line). Every data point represents one individual specimen. See Example 8. Examples
  • Example 1 Preparation of mRNA HA constructs for in vitro and in vivo experiments
  • the target protein was the antigen hemagglutinin of Influenza A virus (A/Netherlands/602/2009(H1 N1 ); Gl:228860929).
  • the C-terminal transmembrane domain (TM) of the protein was removed (amino acids 531 - 566), hereinafter referred to as ⁇ ⁇ ⁇ .
  • a non-heme ferritin of Helicobacter pylori was fused to the C-terminus of ⁇ , separated by a "SGG" spacer sequence, hereinafter referred as ⁇ -SGG-ferritin.
  • a foldon domain of the fibritin/foldon protein of the bacteriophage T4T was fused to the C-terminus of ⁇ , hereinafter referred to as ⁇ - foldon.
  • C3d_P28 was fused to the C-terminus of ⁇ , hereinafter referred to as HA A TM-C3d_P28.
  • a human IgGI Fc domain was fused to the C-terminus of ⁇ , hereinafter referred to as ⁇ -lgG FC.
  • a CD40 ligand domain was fused to a ⁇ , additionally comprising a GCN4pll for trimerization, hereinafter referred to as HAATM-GCN4PI I-CD40L).
  • the fusion constructs used in the present example as well as the control constructs are listed with their respective SEQ ID NOs in Table 1 .
  • DNA sequences encoding the target element ⁇ fused to respective additional elements were prepared and used for subsequent RNA in vitro transcription reactions.
  • the constructs are listed in Table 1 .
  • the DNA sequences were prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence for stabilization. Sequences were introduced into a derived pUC19 vector and modified to comprise stabilizing UTR sequences derived from alpha-globin-3'-UTR (muag (mutated alpha-globin-3'-UTR)), a histone-stem-loop structure, and a stretch of 70 x adenosine at the 3'-terminal end.
  • alpha-globin-3'-UTR alpha-globin-3'-UTR
  • histone-stem-loop structure a stretch of 70 x adenosine at the 3'-terminal end.
  • the respective DNA plasmids were transcribed in vitro using DNA dependent T7 RNA polymerase in the presence of a CAP analog (m7GpppG) and a nucleotide mixture. Subsequently, the in vitro transcribed mRNA was purified using PureMessenger® (CureVac, Tubingen, Germany; WO2008/077592A1 ). The obtained mRNA (naked, unformulated mRNA) was used for in vitro expression analysis.
  • mRNA complexation consisted of a mixture of 50% naked mRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1 .
  • mRNA was complexed with protamine by addition of protamine-Ringer's lactate solution to mRNA. After incubation for 10 minutes, when the complexes were stably generated, naked mRNA was added, and the final concentration of the vaccine was adjusted with Ringer's lactate solution. The obtained formulated mRNA vaccine was used for in vivo experiments.
  • Example 2 Expression of HA constructs in HEK 293T cells and analysis using western blot The aim of these experiments was to analyse the expression of the HA mRNA constructs (see Table 1 ) and to determine the release of the HA protein into the supernatant of transfected HEK 293T cells. All HA mRNA vaccine candidates contained an endogenous secretory signal peptide (N-terminus of the HA protein) that should promote the release from the producing cells into the supernatant. Cell lysates were also analyzed for HA protein expression.
  • HEK 293T cells were seeded in a 24-well plate at a density of 70,000 cells/well in cell culture medium (DMEM complete), 48h prior to transfection. Cells were transfected with and 5 pg naked, unformulated mRNA HA constructs (see Table 1 ) using Lipofectamine 2000 (Invitrogen).
  • transfection supernatants were collected. Additionally, cells were harvested and lysed with RIPA lysis buffer (50 mM Tris-HCI pH 7.4, 1 50 mM NaCI, 1 % TritonX-100, 0.1 % SDS). The respective supernatants and cell lysates were stored at -20 °C. 2.2. Analysis for HA expression using western blot
  • HA was analyzed using a commercially available mouse monoclonal anti influenza A virus H1 N1 specific antibody (Clone 2C10H2, Sino Biological, C) in combination with a goat anti mouse lgG1 IRDye® 800 coupled secondary antibody (LI-COR Biosciences).
  • tubulin was analyzed either i n cel l lysates as a loading control or in supernatants to check for cellular contamination using a rabbit anti ⁇ / ⁇ tubulin antibody (Cell signaling Technology) in combination with a goat anti rabbit IgG IRDye® 680 coupled secondary antibody (LI-COR Biosciences).
  • the approximate protein sizes (without taking post-translational protein modifications into account) are shown in table 2. Western blot results are shown in Figure 1 .
  • HA protein monomers were detected in cell lysates and/or supernatants (see Figure 1 ), showing that mRNA constructs were translated into protein.
  • the band sizes were i n accordance to the expected band sizes.
  • the majority of protein for all four mRNA constructs was detected in the respective supernatants (see Figure 1 B). Since no tubulin protein was detectable in the analyzed supernatants (data not shown), the presence of HA protein was considered to be mediated by secretion triggered by the endogenous secretory signal peptide and not via release by cell death associated with the transfection method.
  • all tested mRNA constructs were translated and secreted in HEK 293T cells.
  • mice Female BALB/c mice were injected intramuscularly (i.m.) with formulated mRNAs vaccines encoding HA protein constructs with doses indicated in Table 3. As a negative control, one group of mice was vaccinated with buffer (ringer lactate, RiLa). All animals received boost injections on day 21 . Blood samples were collected on day 21 and 28 for the analysis of the immune response in the effector phase (see Examples 4-5) and additionally on day 35 and 49 for the analysis of the immune response in the memory phase (see Examples 6-7).
  • Example 4 ELISA analysis of an antigen specific humoral immune response in the effector phase
  • the aim of this experiment was to assess the antigen specific humoral immune response in vaccinated mice for the used mRNA vaccines and to compare the detected immune response evoked by HA fusion constructs (with additional element) with the immune response evoked by the target HA antigen without additional element.
  • HA protein specific IgGI and lgG2a antibodies were detected by ELISA using sera obtained at day 21 and day 28 (effector phase).
  • Example 5 Hemagglutination inhibition assay to determine virus neutralizing titers in the effector phase
  • the aim of this experiment was to determine virus neutralizing titers in the collected mice sera (see Example 3) and to compare virus neutralizing titers of mice vaccinated with HA fusion constructs to the titers of mice vaccinated with the target HA antigen without additional element.
  • Hemagglutination inhibition assay (HI) Hemagglutination inhibition assay
  • Example 6 ELISA analysis of an antigen specific humoral immune response in the memory phase
  • ELISA was performed according to example 4.
  • Vaccination was performed according to example 3.
  • sera obtained at day 35 (two week after boost vaccination) and day 49 (four weeks after boost vaccination) were used. The results are shown in Figure 4.
  • the HI assay was performed according to example 5.
  • Vaccination was performed according to example 3.
  • sera obtained at day 49 (four weeks after boost vaccination) was used. The results are shown in Figure 5.
  • Example 8 Hemagglutination inhibition assay to determine functional antibody titers
  • the vaccination schemes as well as the used concentrations are provided in Table 4. The results are shown in Figure 6. Table 4: Vaccination regimen for indicated animal groups
  • the target protein is the mice EPO protein (MmEPO; Gl:
  • the DNA sequences encoding the target EPO (SEQ ID NO: 1 771 ) fused to respective additional half life extension elements are prepared by modifying the wild type encoding DNA sequences by introducing a GC-optimized sequence and/or codon optimized sequence for stabilization and optimized expression. Sequences were introduced into a vector and modified to additionally comprise stabilizing UTR sequences (3' UTR and 5' UTR), a histone-stem-loop structure, a poly-A stretch, and a poly-C stretch at the 3'-terminal end.
  • the DNA constructs are used as templates for subsequent RNA in vitro transcription reactions (see Example ⁇ ). Subsequently, the in vitro transcribed mRNA is purified using PureMessenger® (CureVac, Tubingen, Germany; WO2008/077592A1 ).
  • HEK 293T cells and HeLa cells are seeded in a 24-well plate at a density of 70,000 cells/well in cell culture medium 48h prior to transfection.
  • Cells are transfected with 5 pg naked, unformulated mRNA EPO constructs (see Table 4) using Lipofectamine 2000 (Invitrogen).
  • As a control full length EPO mRNA construct is used (without half-life extending element).
  • 24 hours post transfection cell culture supernatants are collected. Additionally, cells are harvested and lysed with RIPA lysis buffer (50 mM Tris-HCI pH 7.4, 1 50 mM NaCI, 1 % TritonX-100, 0.1 % SDS) or harvested using SDS lysis buffer.
  • RIPA lysis buffer 50 mM Tris-HCI pH 7.4, 1 50 mM NaCI, 1 % TritonX-100, 0.1 % SDS
  • An SDS-PAGE is performed with supernatants and whole cell lysates from all samples with Mini-PROTEAN® TGX Precast Mini Gels 4 - 20 % (Gradient gel; Bio-Rad). Untransfected cells are used as a negative control.
  • the blotting on a nitrocellulose membrane is performed for 2h in the presence of a blotting buffer. After blocking the membrane in a respective buffer, antibody incubation (primary and secondary antibodies) and signal detection (Ll- COR measurement) is performed. The presence of EPO is analyzed using a commercially available anti EPO specific antibody in combination with a suitable IgGI IRDye® 800 coupled secondary antibody (LI-COR Biosciences).
  • EPO levels in the culture medium are quantitatively measured 24 hours post transfection using a commercially available mouse EPO ELISA kit (R&D Systems, Wiesbaden, Germany).
  • the constructs showing suitable expression and secretion characteristics are used in in vivo experiments. 9.3. In vivo characterization of TransIT formulated EPO constructs
  • mRNA encoding said fusion proteins is formulated for in vivo application using TransIT and injected
  • EPO protein and TransiT formulated EPO mRNA are used as control. 6 hours, 1 day, 4 days and 7 days after injection, a few microliters of blood are collected, heparinized, and centrifuged. EPO levels in the supernatant are measured using a mouse EPO ELISA kit (R&D Systems). In addition, reticulocytes are analysed using a commercially avai lable Retic-COUNT kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions. Stained cells are analyzed on a FACS Canto (BD Biosciences). Reticulocyte levels are given as percentage of total red blood cells.

Abstract

La présente invention concerne des molécules d'acide nucléique optimisées, des procédés d'optimisation de molécules d'acide nucléique et des utilisations de molécules d'acide nucléique optimisées. L'invention concerne un principe de construction modulaire permettant de générer un acide nucléique, notamment un ARN messager, qui est conçu pour une application donnée. Les molécules d'acide nucléique selon la présente invention peuvent être obtenues par la combinaison polyvalente de multiples modules au niveau de l'acide nucléique. Un tel acide nucléique, par exemple un ARNm, peut être conçu en associant un ou plusieurs modules, comprenant (i) une fraction d'acide nucléique codant un polypeptide d'intérêt (par exemple une protéine produisant éventuellement un résultat thérapeutique) et (ii) au moins une autre fraction d'acide nucléique codant ou non codant, par exemple une fraction choisie parmi des fractions d'acide nucléique codant un élément polypeptidique, par exemple un peptide signal de sécrétion (SSP), un élément de multimérisation (dimérisation, trimérisation, tétramérisation et oligomérisation), un élément formant une particule du type viral (VLP), un élément transmembranaire, un élément ciblant les cellules dendritiques, un élément adjuvant immunologique, un élément favorisant la présentation de l'antigène ; un peptide 2A ; un élément de liaison peptidique, des éléments qui prolongent la demi-vie de protéines et/ou toute autre polypeptide ou protéine. Des fragments d'acide nucléique non codant peuvent être choisis par exemple dans le groupe comprenant 3'-UTR, 5'-UTR, l'élément IRES, le fragment ARNmi, une boucle histone, une séquence poly(C), un signal de polyadénylation, une séquence poly(A). La molécule d'acide nucléique optimisée peut en outre être caractérisée par la présence d'au moins un nucléoside modifié. La polyvalence de la présente invention permet la conception rationnelle d'une grande variété de molécules d'acide nucléique différentes ayant des propriétés souhaitées.
PCT/EP2016/077145 2015-11-09 2016-11-09 Molécules d'acide nucléique optimisées WO2017081082A2 (fr)

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US20200399322A1 (en) 2020-12-24
US20180312545A1 (en) 2018-11-01

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